Human neutrophil gelatinase-associated lipocalin (hngal) muteins that bind hepcidin and nucleic acid encoding such

ABSTRACT

The present invention relates to novel, specific-binding therapeutic and/or diagnostic proteins directed against Hepcidin, which proteins preferably are muteins of lipocalin protein. The invention also relates to nucleic acid molecules encoding such proteins and to methods for generation and use of such proteins and nucleic acid molecules. Accordingly, the invention also is directed to pharmaceutical and/or diagnostic compositions comprising such a lipocalin proteins, including uses of these proteins.

The present invention relates to novel, specific-binding therapeuticand/or diagnostic proteins directed against Hepcidin, which proteinspreferably are muteins of a lipocalin protein. The invention alsorelates to nucleic acid molecules encoding such proteins and to methodsfor generation and use of such proteins and nucleic acid molecules.Accordingly, the invention also is directed to pharmaceutical and/ordiagnostic compositions comprising such a lipocalin proteins, includinguses of these proteins.

BACKGROUND

Hepcidin, a peptide hormone typically existing in two forms made ofeither 20 or 25 amino acids, is expressed and secreted by a number ofcells in response to iron loading and inflammation. Hepcidin is producedpredominantly in hepatocytes of the liver, plays a central role in theregulation of iron homeostasis, acts as an antimicrobial peptide and isdirectly or indirectly involved in the development of mostiron-deficiency/overload syndromes. A major action of hepcidin is tointernalize and degrade the iron exporter ferroportin, which isexpressed on all iron-exporting cells. Hepcidin directly binds toferroportin. A high hepcidin level thus leads to the suppression ofintestinal iron absorption and iron release from macrophages andhepatocytes, while a low concentration of hepcidin leads to accelerationof iron release from these cells.

Hepcidin is also suspected to play role in pathogenesis of anemia ofinflammation and iron-deficiency anemia. Anemia of inflammation, alsoknown as anemia of chronic disease (ACD) or anemia of chronic disorders,currently is the most frequent anemia among hospitalized patients and acommon syndrome complicating many infectious, non-infectiousinflammatory and neoplastic disorders. ACD is a normocytic, normochromicanemia characterized by decreased iron and iron-binding capacity(transferrin), increased ferritin and the presence of iron in bonemarrow macrophages, indicating impaired mobilization of iron from itsstores. While in anemia of inflammation hepcidin levels are increased,in iron-deficiency anemia low hepcidin levels are found. Hence, hepcidincould be used as a marker to distinguish these diseases. Hepcidin mayalso be a useful marker for screening, prognosis and monitoringhereditary hemochromatosis and iron loading anemias. Hepcidin levels mayfurther be useful in monitoring EPO treatment and predicting a responseto EPO.

Methods of isolating, analyzing and quantifying hepcidin as well asagents for the treatment of diseases and conditions associated withhepcidin have been described in international patent applications WO2008/011158, WO 2008/097461, WO 2009/094551A1, WO 2009/139822, WO20091058797 and WO 2010/017070. However, no hepcidin-binding proteinhaving the features attendant to the proteins provided by presentinvention has been previously described.

SUMMARY OF THE INVENTION

One embodiment of the current relates to a lipocalin mutein that iscapable of binding hepcidin with an affinity measured by a K_(D) ofabout 10 nM or lower. More preferably, the lipocalins can have anaffinity measured by a K_(D) of about 1 nM or lower. In anotherembodiment, the lipocalin mutein is capable of neutralizing thebioactivity of human hepcidin-25, preferably with an IC50 value of about80 nM or lower as determined by a cell-based assay for hepcidin-inducedinternalization and degradation of ferroportin.

In particular embodiments, a lipocalin mutein according to the currentinvention comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-14. In another embodiment, the mutein has atleast 75% identity to the sequence of a wild-type human lipocalin,including human Lipocalin 2.

In another embodiment, the mutein of the current invention is conjugatedto a compound selected from the group consisting of an organic molecule,an enzyme label, a radioactive label, a colored label, a fluorescentlabel, a chromogenic label, a luminescent label, a hapten, digoxigenin,biotin, a cytostatic agent, a toxins, a metal complexe, a metal, andcolloidal gold. The mutein can be fused at its N-terminus and/or itsC-terminus to a fusion partner which is a protein, a protein domain, ora peptide.

In another embodiment, the mutein is conjugated to a compound thatextends the serum half-life of the mutein. More preferably, the muteinis conjugated to a compound selected from the group consisting of apolyalkylene glycol molecule, a hydroethylstarch, an Fc part of animmunoglubolin, a C_(H)3 domain of an immoglobulin, a C_(H)4 domain ofan immunoglubolin, an albumin binding peptide, and an albumin bindingprotein.

In another embodiment, the mutein of the current invention is anantagonist of a Hepcidin. The hepcidin can be mature human Hepcidin.

In another embodiment, the current invention relates to a nucleic acidmolecule comprising a nucleotide sequence encoding a mutein of thecurrent invention.

In another embodiment, the lipocalin mutein of the current invention isselected from the group consisting of muteins of retinol-binding protein(RBP), bilin-binding protein (BBP), apolipoprotein D (APO D), neutrophilgelatinase associated lipocalin (NGAL), tear lipocalin (TLPC),α₂-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), vonEbners gland protein 1 (VEGP 1), von Ebners gland protein 2 (VEGP 2),and Major allergen Can f1 precursor (ALL-1). In related embodiments, thelipocalin mutein is selected from the group consisting of humanneutrophil gelatinase associated lipocalin (hNGAL), human tear lipocalin(hTLPC), human apolipoprotein D (APO D) and the bilin-binding protein ofPieris brassicae.

In another embodiment, the invention relates to a lipocalin mutein whichprevents human hepcidin-25 induced reduction of serum iron levels in asubject.

The invention also includes a method of treating a disease or disorderassociated with an altered level of a Hepcidin, the method comprisingadministering a pharmaceutical composition containing a mutein asdescribed herein to a subject in need thereof. In related embodiments,the disease or disorder involves a disorder of iron homeostasis or aninflammatory condition associated with an elevated level of hepcidin.

DESCRIPTION OF FIGURES

FIG. 1 illustrates the PCR assembly strategy for the simultaneous randommutagenesis of the 20 amino acid positions 36, 40, 41, 49, 52, 68, 70,72, 73, 77, 79 81, 968, 100, 103, 106, 125, 127, 132, and 134(underlined and numbered) in the amino acid sequence of the mature Lcn2. These 20 positions were divided into four sequence subsets. Forrandomization of the amino acids in each subset an oligodeoxynucleotidewas synthesized (SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19) wherein NNK mixtures of the nucleotides were employed at the mutatedcodons. N means a mixture of all four bases A, C, G, and T while K meansa mixture of only the two bases G and T; hence such a triplet encodesall 20 natural amino acids as well as the amber stop codon TAG, which istranslated as glutamine in the E. coli supE-strains XL1-blue (Bullock etal., BioTechniques 5 (1987), 376-378) or TG1 (Sambrook et al., MolecularCloning. A Laboratory Manual (1989), Cold Spring Harbor Press) that wereused for phagemid production and gene expression. Four additionaloligodeoxynucleotides (SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23) with fixed nucleotide sequences corresponding to thenon-coding strand (written below the DNA double strand sequence in 3′-5′direction) and filling the gaps between the aforementionedoligodeoxynucleotides were also used in the assembly reaction. Twoshorter flanking oligodeoxynucleotides (SEQ ID NO. 24 and SEQ ID NO:25), which were added in excess and carried biotin groups, served asprimers for the PCR amplification of the assembled, entirely syntheticgene fragment. The two flanking primers each encompassed a BstXIrestriction site (CCANNNNNNTGG) giving rise to mutually non-compatibleoverhangs upon enzyme digestion. This special arrangement of restrictionsites enabled a particularly efficient ligation and cloning of thesynthetic gene. Substitution of the amino acid Gln28 to His with respectto the original Lcn2 sequence was necessary to introduce the first BstXIsite, while the second one naturally occurs in the cDNA of Lcn2.Furthermore, the unpaired residue Cys87 was replaced by Ser during thegene assembly. After one pot PCR the resulting gene fragment wasinserted into a vector providing the missing parts of the Lcn2structural gene. This illustration also depicts two short primers (SEQID NO: 32 and SEQ ID NO: 33) upstream and downstream, respectively, ofthe cassette flanked by the two BstXI restriction sites, which servedfor double stranded DNA sequencing.

FIG. 2 illustrates the nucleotide sequence of a library of syntheticLcn2 genes (only the central cassette flanked by the two BstXIrestriction sites, as in FIG. 1, is shown). This gene fragment wasprepared by Sloning BioTechnology GmbH. Compared with the DNA librarydescribed in FIG. 1 there are two differences. First, whenever possible,codons optimized for E. coli expression were used throughout for thenon-mutated amino acid positions. Second, a mixture of 19 differenttriplets (GAC, TTC, CTG, CAC, AAT, AGC, ACC, GCA. ATG, CCT, GTT, TGG,GAG, CAA, ATC, GGA, CGT, GCA, TAC), each encoding a different amino acidexcept Cys, was employed at the 20 randomized positions, which areidentical to the ones depicted in FIG. 1. Numbering of amino acidscorresponds here to an internal scheme employed by Sloning BioTechnologyGmbH, whereby Gly No 1 is the first amino acid codon directly followingthe upstream BstX1 restriction site.

FIG. 3A depicts an alignment of certain amino acid sequences ofhHepcidin-specific, NGAL-based lipocalin muteins in comparison with thepolypeptide sequence of wildtype NGAL lipocalin. The NGAL-derived,hepdicin binding muteins comprise residues 1 to 178, meaning they havethe length of the mature wildtype proteins. Residues 179 to 186 are thesequence of a streptavidin binding tag, Strep-Tag™, used in theisolation of generated muteins.

FIG. 3B depicts an alignment of certain amino acid sequences ofhHepcidin-specific, NGAL-based lipocalin muteins in comparison with thepolypeptide sequence of wildtype NGAL lipocalin. The NGAL-derived,hepdicin binding muteins comprise residues 1 to 178, meaning they havethe length of the mature wildtype proteins. Residues 179 to 186 are thesequence of a streptavidin binding tag, Strep-Tag™, used in theisolation of generated muteins.

FIG. 4A depicts the amino acid sequence of the lipocalin mutein of SEQID NO: 1 fused, via a linker (greyish bold italic) to an ABD domain(bold) and a streptavidin binding tag, Strep-Tag™ (italic) (SEQ ID NO:15).

FIG. 4B depicts the amino acid sequence of the lipocalin hNGAL, asencoded by the vector phNGAL 98, fused to a streptavidin binding tag,the Strep-Tag™ (italic) and an N-terminal T7 tag (bold italic) (SEQ IDNO. 34). This polypeptide is encoded by phNGAL 101.

FIG. 5 shows the results of a direct ELISA of selected Lcn2 muteins.

FIG. 6 depicts the results of a competitive binding assay of selectedLcn2 muteins.

FIG. 7 depicts the affinities of selected muteins for human andcynomolgus Hepcidin-25 as determined by surface-plasmon-resonance (SPR).

FIG. 8 depicts the in vitro neutralization activity of anti-Hepcidin-25lipocalin muteins.

FIG. 9 demonstrates that a lipocalin mutein directed against hepcidinneutralizes human hepcidin injected into mice.

FIG. 10 depicts pharmacokinetic parameters for SEQ ID NO: 14-PEG and SEQID NO: 1-ABD (equal to SEQ ID NO: 15).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the present invention relates to novel, specific-bindingproteins directed against or specific for hepcidin. Proteins of theinvention may be used for therapeutic and/or diagnostic purposes. Asused herein, a protein of the invention “specifically binds” a target(here, hepcidin) if it is able to discriminate between that target andone or more reference targets, since binding specificity is not anabsolute, but a relative property. “Specific binding” can be determined,for example, in accordance with Western blots, ELISA-, RIA-, ECL-,IRMA-tests, FACS, IHC and peptide scans.

Proteins of the invention, which are directed against or specific forhepcidin, include any number of specific-binding protein muteins thatare based on a defined protein scaffold. As used herein, a “mutein,” a“mutated” entity (whether protein or nucleic acid) or “mutant” refers tothe exchange, deletion, or insertion of one or more nucleotides or aminoacids, respectively, compared to the naturally occurring (wild-type)nucleic acid or protein “reference” scaffold.

A protein of the invention can be a mutein of a lipocalin, preferably alipocalin selected from the group consisting of retinol-binding protein(RBP), bilin-binding protein (BBP), apolipoprotein D (APO D), neutrophilgelatinase associated lipocalin (NGAL), tear lipocalin (TLPC),α₂-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), vonEbners gland protein 1 (VEGP 1), von Ebners gland protein 2 (VEGP 2),and Major allergen Can fi precursor (ALL-1). As used herein, a“lipocalin” is defined as monomeric protein of approximately 18-20 kDAin weight, having a cylindrical β-pleated sheet supersecondarystructural region comprising a plurality of (preferably eight) β-strandsconnected pair-wise by a plurality of (preferably four) loops at one endto define thereby a binding pocket. It is the diversity of the loops inthe otherwise rigid lipocalin scaffold that gives rise to a variety ofdifferent binding modes among the lipocalin family members, each capableof accommodating targets of different size, shape, and chemicalcharacter (reviewed, e.g., in Flower, D. R. (1996), supra; Flower, D. R.et al. (2000), supra or Skerra, A. (2000) Biochim. Biophys. Acta 1482,337-350). Indeed, the lipocalin family of proteins have naturallyevolved to bind a wide spectrum of ligands, sharing unusually low levelsof overall sequence conservation (often with sequence identities of lessthan 20%) yet retaining a highly conserved overall folding pattern. Thecorrespondence between positions in various lipocalins is well known toone of skill in the art. See, for example, U.S. Pat. No. 7,250,297.

In a preferred embodiment, a protein of the invention is a mutein ofLipocalin 2 (Lcn 2; also known as human neutrophil gelatinase-associatedlipocalin, hNGAL, or as siderocalin). The term “human neutrophilgelatinase-associated lipocalin” or “hNGAL” or “lipocalin 2” or “Lcn2”as used herein to refer to the mature hNGAL with the SWISS-PROT/UniProtData Bank Accession Number P80188 or the mature hNGAL shown in SEQ IDNO:35. The mature form of this protein has amino acids 21 to 198 of thecomplete sequence, since a signal peptide of amino acids 1-20 is cleavedoff. The protein further has a disulfide bond formed between the aminoacid residues at positions 76 and 175 of the mature protein.

In a more preferred embodiment, the invention relates to a lipocalinmutein having a cylindrical β-pleated sheet supersecondary structuralregion comprising eight β-strands connected pair-wise by four loops atone end to define thereby a binding pocket, wherein at least one aminoacid of each of at least three of said four loops has been mutated andwherein said lipocalin is effective to bind a hepcidin as givennon-natural target with detectable affinity. Preferably, said lipocalinmutein has one or more such as 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acid relacements at a positioncorresponding to position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79,81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of the linearpolypeptide sequence of NGAL.

In this context, the inventors identified a specific group of Lipocalin2 muteins with mutations at specific positions which show detectableaffinity as well as specificity for Hepcidin. Suitable amino acidpositions for mutation include sequence positions 96, 100, and 106, ofthe linear polypeptide sequence of human Lipocalin 2. The presentinvention also relates to nucleic acids encoding these proteins.

Other protein scaffolds that can be engineered in accordance with thepresent invention to provide protein muteins that bind hepcidin withdetectable affinity include: an EGF-like domain, a Kringle-domain, afibronectin type I domain, a fibronectin type II domain, a fibronectintype III domain, a PAN domain, a G1a domain, a SRCR domain, aKunitz/Bovine pancreatic trypsin Inhibitor domain, tendamistat, aKazal-type serine protease inhibitor domain, a Trefoil (P-type) domain,a von Willebrand factor type C domain, an Anaphylatoxin-like domain, aCUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain,a Sushi domain, a Link domain, a Thrombospondin type I domain, animmunoglobulin domain or a an immunoglobulin-like domain (for example,domain antibodies or camel heavy chain antibodies), a C-type lectindomain, a MAM domain, a von Willebrand factor type A domain, aSomatomedin B domain, a WAP-type four disulfide core domain, a F5/8 typeC domain, a Hemopexin domain, an SH2 domain, an SH3 domain, aLaminin-type EGF-like domain, a C2 domain, “Kappabodies” (Ill, et al.“Design and construction of a hybrid immunoglobulin domain withproperties of both heavy and light chain variable regions” Protein Eng10:949-57 (1997)), “Minibodies” (Martin et al. The affinity-selection ofa minibody polypeptide inhibitor of human interleukin-6′ EMBO J13-5303-9 (1994)), “Diabodies” (Holliger et al. “Diabodies”: smallbivalent and bispecific antibody fragments' PNAS USA 90:6444-6448(1993)), “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new moleculardesign for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992), ananobody, an adnectin, a tetranectin, a microbody, an affilin, anaffibody an ankyrin, a crystallin, a knottin, ubiquitin, a zinc-fingerprotein, an autofluorescent protein, an ankyrin or ankyrin repeatprotein or a leucine-rich repeat protein, an avimer (Silverman, Lu Q.Bakker A, To W, Duguay A, Alba B M, Smith R, Rivas A, Li P, Le H.Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M, Kolkman J,Stemmer W P 2005, Nat Biotech, December; 23(12):1556-61. E-Publicationin Nat Biotech. 2005 Nov. 20 edition); as well as multivalent avimerproteins evolved by exon shuffling of a family of human receptor domainsas also described in Silverman J, Lu Q, Bakker A, To W, Duguay A, Alba BM, Smith R. Rivas A, Li P, Le H, Whitehorn E, Moore K W, Swimmer C,Perlroth V, Vogt M. Kolkman J, Stemmer W P, Nat Biotech, December:23(12):1556-61, E-Publication in Nat. Biotechnology. 2005 Nov. 20edition).

A protein of the invention may include the wild type (natural) aminoacid sequence of the “parental” protein scaffold (such as a lipocalin)outside the mutated amino acid sequence positions; alternatively, alipocalin mutein may also contain amino acid mutations outside thesequence positions subjected to mutagenesis that do not interfere withthe binding activity and the folding of the mutein. Such mutations canbe accomplished on a DNA level using established standard methods(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Possible alterations of the amino acid sequence are insertions ordeletions as well as amino acid substitutions.

Such substitutions may be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine: 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan One the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of a parental protein scaffold, where thesedeletions or insertion result in a stable folded/functional mutein,which can be readily tested by the skilled worker.

The skilled worker will appreciate methods useful to prepare proteinmuteins contemplated by the present invention but whose protein ornucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence include. e.g.,directed mutagenesis of single amino acid positions in order to simplifysubcloning of a mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for a given target. Furthermore, mutations can beintroduced to modulate certain characteristics of the mutein such as toimprove folding stability, serum stability, protein resistance or watersolubility or to reduce aggregation tendency, if necessary. For example,naturally occurring cysteine residues may be mutated to other aminoacids to prevent disulphide bridge formation.

Accordingly, the invention also includes functional variants of proteinsdisclosed herein, which have a threshold sequence identity or sequencehomology to a reference protein. By “identity” or “sequence identity” ismeant a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent invention means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the invention with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Percent identity isdetermined by dividing the number of identical residues by the totalnumber of residues and multiplying the product by 100. The term“homology” is used herein in its usual meaning and includes identicalamino acids as well as amino acids which are regarded to be conservativesubstitutions (for example, exchange of a glutamate residue by anaspartate residue) at equivalent positions in the linear amino acidsequence of two proteins. Most preferred, the amino acid sequence shownin SEQ ID NO:35 is preferred as a “reference sequence”. SEQ ID NO:35shows the mature hNGAL. The term “reference sequence” and “wild typesequence” (of NGAL) is used interchangeably herein. Alternatively, theamino acid sequence with the SWISS-PROT/UniProt Data Bank AccessionNumber P80188 can be used as reference sequence.

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1, cutoff value set to 10⁻³) including the propeptidesequences, preferably using the wild type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

It is also possible to deliberately mutate other amino acid sequencepositions to cysteine in order to introduce new reactive groups, forexample, for the conjugation to other compounds, such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins,or for the formation of non-naturally occurring disulphide linkages.With respect to a mutein of human Lipocalin 2, exemplary possibilitiesof such a mutation to introduce a cysteine residue into the amino acidsequence of a lipocalin including human Lipocalin 2 mutein to includethe introduction of a cysteine (Cys) residue at at least at one of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGAL.In some embodiments where a human Lipocalin 2 mutein of the inventionhas a sequence in which, in comparison to the sequence of theSWISS-PROT/UniProt Data Bank Accession Number P80188, a cysteine hasbeen replaced by another amino acid residue, the corresponding cysteinemay be reintroduced into the sequence. As an illustrative example, acysteine residue at amino acid position 87 may be introduced in such acase by reverting to a cysteine as originally present in the sequence ofSWISS-PROT accession No P80188. The generated thiol moiety at the sideof any of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145,143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, forexample, in order to increase the serum half-life of a respective humanLipocalin 2 mutein.

The term “position” when used in accordance with the invention means theposition of either an amino acid within an amino acid sequence depictedherein or the position of a nucleotide within a nucleic acid sequencedepicted herein. The term “corresponding” as used herein also includesthat a position is not only determined by the number of the precedingnucleotides/amino acids. Accordingly, the position of a given amino acidin accordance with the invention which may be substituted may very dueto deletion or addition of amino acids elsewhere in a (mutant orwild-type) lipocalin. Similarly, the position of a given nucleotide inaccordance with the present invention which may be substituted may varydue to deletions or additional nucleotides elsewhere in a mutein or wildtype lipocalin 5′-untranslated region (UTR) including the promoterand/or any other regulatory sequences or gene (including exons andintrons).

Thus, under a “corresponding position” in accordance with the inventionit is preferably to be understood that nucleotides/amino acids maydiffer in the indicated number but may still have similar neighboringnucleotides/amino acids. Said nucleotides/amino acids which may beexchanged, deleted or added are also comprised by the term“corresponding position”. When used herein “at a position correspondingto a position” a position in a “query” amino acid (or nucleotide)sequence is meant that corresponds to a position in a “subject” aminoacid (or nucleotide) sequence.

Specifically, in order to determine whether a nucleotide residue oramino acid residue of the amino acid sequence of a lipocalin differentfrom a NGAL lipocalin mutein of the invention corresponds to a certainposition in the nucleotide sequence or the amino acid sequence of a NGALlipocalin mutein as described, in particular any of SEQ ID NOs: 1-14 orthat having one or more amino acid substitutions such as 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 at position 36,40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127,132, and/or 134 of the linear polypeptide sequence of NGAL, a skilledartisan can use means and methods well-known in the art, e.g.,alignments, either manually or by using computer programs such asBLAST2.0, which stands for Basic Local Alignment Search Tool or ClustalWor any other suitable program which is suitable to generate sequencealignments. Accordingly, a lipocalin mutein of any of SEQ ID Nos: 1-14or that having one or more amino acid substitutions such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 at position 36,40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127,132, and/or 134 or any other position described herein of the linearpolypeptide sequence of NGAL can serve as “subject sequence”, while theamino acid sequence of a lipocalin different from NGAL serves as “querysequence”.

Given the above, a skilled artisan is thus readily in a position todetermine which amino acid position mutated in Lcn2 as described hereincorresponds to an amino acid of a scaffold other than Lcn2, preferablysuch as one of those described herein. Specifically, a skilled artisancan align the amino acid sequence of a mutein as described herein, inparticular a NGAL mutein (or anticalin) of the invention with the aminoacid sequence of a different lipocalin to determine which amino acid(s)of said mutein correspond(s) to the respective amino acid(s) of theamino acid sequence of said different lipocalin. More specifically, askilled artisan can thus determine which amino acid of the amino acidsequence of said different lipocalin corresponds to the amino acid atposition(s) 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100,103, 106, 125, 127, 132, and/or 134 or to an amino acid at any otherposition as described herein of the linear polypeptide sequence of NGAL.

Proteins of the invention, which are directed against or specific forhepcidin, include any number of specific-binding protein muteins thatare based on a defined protein scaffold. Preferably, the scaffold ishNGAL. As used herein, a “mutein,” a “mutated” entity (whether proteinor nucleic acid) or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, respectivelycompared to the naturally occurring (wild-type) nucleic acid or protein“reference” scaffold. Preferably, the number of nucleotides or aminoacids, respectively, that is exchanged, deleted or inserted is 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moresuch as 25, 30, 35, 40, 45 or 50. However, it is preferred that a muteinof the invention is still capable of binding hepcidin.

In some embodiments, a protein according to the invention binds ahepcidin with a K_(D) of 100 μM or less, including 5 μM or less, about500 nM, about 200 nM or less, 100 nM or less, 1 nM or less, or 0.1 nM orless. A protein of the invention may specifically bind one or morecontinuous, discontinuous or conformation epitope(s) of the mature,folded bioactive form of a hepcidin.

A protein of the invention is able to bind a hepcidin with detectableaffinity, i.e. with a dissociation constant of at least 200 nM, i.e.K_(D) of about 200 nM or less. In some embodiments, a protein of theinvention binds a hepcidin with a dissociation constant of at leastabout 100 nM, about 50 nM, about 25 nM, about 15 nM, about 5 nM, about 2nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.05 nM or evenless. A protein of the invention preferably binds to a mature humanhepcidin molecule with an affinity by a K_(D) of about 10 nM orstronger. Binding affinities have been found by the present inventors tooften be of a K_(D) below about 1 nM and, in some cases, about 0.1 nMand below.

The binding affinity of a protein of the invention (e.g. a mutein of alipocalin) to a selected target (in the present case, hepcidin), can bemeasured (and thereby K_(D) values of a mutein-ligand complex bedetermined) by a multitude of methods known to those skilled in the art.Such methods include, but are not limited to, fluorescence titration,competition ELISA, calorimetric methods, such as isothermal titrationcalorimetry (ITC), and surface plasmon resonance (BIAcore). Such methodsare well established in the art and examples thereof are also detailedbelow.

The amino acid sequence of a protein of the invention may have a highsequence identity to mature human Lipocalin 2 or other lipocalins. Inthis context, a protein of the invention may have at least 70%, at least75%, at least 80%, at least 82%, at least 85%, at least 87%, at least90% identity, including at least 95% identity to a protein selected fromthe group consisting of the sequence of SEQ ID NOS: 1-14. It ispreferred that a structural homolog has still an amino acid replacementat one or more such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 positions corresponding to position 36, 40, 41, 49,52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and/or134 of the linear polypeptide sequence of NGAL.

The invention also includes structural homologues of the proteinsselected from the group consisting of the sequence of SEQ ID NOS: 1-14,which have an amino acid sequence homology or sequence identity of morethan about 60%, preferably more than 65%, more than 70%, more than 75%,more than 80%, more than 85%, more than 90%, more than 92% and mostpreferably more than 95% in relation thereto. It is preferred that astructural homolog has still an amino acid replacement at one or moresuch as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 positions corresponding to position 36, 40, 41, 49, 52, 68, 70,72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of thelinear polypeptide sequence of NGAL.

The term “hepcidin” refers to the protein also termed liver-expressedantimicrobial peptide 1 or putative liver tumor regressor, of which thehuman form has the UniProtKB/Swiss-Prot accession number P81172. On ageneral basis the term “hepcidin” refers to any form of the hepcidinprotein known to be present in vertebrate species, including in mammals.The human unprocessed protein has a length of 84 amino acids and isencoded by the gene “HAMP,” also known as “HEPC” or “LEAP1.” It iscleaved into two chains, which are herein also included in the term“Hepcidin.” These two chains are of amino acids 60-84, which isHepcidin-25 (Hepc25) and of amino acids 65-84, which is Hepcidin-20(Hepc20). Hepcidin-25 is arranged in the form of a bent hairpin,stabilized by four disulfide bonds Natural variants also included in theterm “hepcidin” have, for example, the amino acid replacement 59 R→G(VAR_(—)0425129); the amino acid replacement 70 C→R (VAR_(—)042513); theamino acid replacement 71 G→D (VAR_(—)026648) or the amino acidreplacement 78 C→Y (VAR_(—)042514). A further natural variant isHepcidin-22, another N-terminally truncated isoform (besides Hecidin-20)of Hepcidin-25.

The term “mature hepcidin” as used herein refers to any mature,bioactive form of the hepcidin protein expressed in a vertebrate such asa mammal. The term “human hepcidin” refers to any form of the hepcidinprotein present in humans. The expression “human hepcidin-25” refers tothe mature form of human hepcidin with the amino acid sequence asdepicted in SEQ ID NO: 28. In the present invention lipocalin muteinsare provided that are able to bind each given form of hepcidin includingproteolytic fragments thereof, regardless of whether the respectivehepcidin molecule displays biological/physiological activity. Thus, thehepcidin molecule may only be present in a biological sample, withouthaving any measurable physiological relevance. See, for example,Hepcidin-22 that so far has only been detected in urine found in urineand that so far is assumed to merely be a urinary degradation product ofHepcidin-25 (reviewed in Kemna et al., Haematologica, 2008 January;93:(1)90-97). A mutein of the invention may of course also bindphysiological active species such as the mature, bioactive Hepcidin-25.Accordingly, a mutein of the invention may be used as diagnostic and/orpharmaceutical, depending on the hepcidin form chosen to be recognized.

In line with the above, a protein of the invention preferably acts as anantagonist of a hepcidin molecule. In some embodiments, a protein of theinvention (e.g., a human Lipocalin 2 mutein) may act as an antagonist ofa hepcidin molecule by inhibiting the ability of the hepcidin moleculeto bind to or otherwise interact with ferroportin. The hepcidin may be amature human hepcidin format such as hepcidin-25 or hepcidin-20. Bindingof a mature hepcidin to ferroportin leads to internalization anddegradation of ferroportin, standard processes of a protein with a cellsurface/membrane location.

In yet another aspect, the present invention includes various lipocalinmuteins, including muteins of human Lipocalin 2 that specifically bindhepcidin. In this sense, hepcidin can be regarded a non-natural ligandof wild type human Lipocalin 2, where “non-natural ligand” refers to acompound that does not bind to wildtype lipocalins, including humanLipocalin 2 under physiological conditions. By engineering wildtypelipocalins such as human Lipocalin 2 with mutations at certainpositions, the present inventors have demonstrated that high affinityand high specificity for a non-natural ligand is possible. In one aspectat least at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, and/or 20 nucleotide triplet(s) encoding for any of the sequencepositions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103,106, 125, 127, 132, and/or 134 of the linear polypeptide sequence ofhLcn2, or other parallel sites on lipocalins, a random mutagenesis canbe carried out by allowing substitution at this positions by a subset ofnucleotide triplets.

The amino acid replacements in the lipocalin muteins of the invention asdescribed herein are preferably within one, two, three or four loopregions of a lipocalin, preferably hNGAL. The loop regions are frompositions 33 to 54 (loop 1), 686 to 83 (loop 2), 94 to 106 (loop 3), and123 to 136 (loop 4) of hNGAL 24-36, 53-66, 79-84, and 103-110

Further, the lipocalins can be used to generate muteins that have amutated amino acid residue at any one or more, including at least at anytwo or all three, of the sequence positions of the sequence positionscorresponding to the sequence positions 96, 100 and 106 of the linearpolypeptide sequence of a mature human Lipocalin 2. A substitution atsequence position 96 may for example be a substitution Asn 96→Arg, Asp,Gln, Gly, Lys, Ser, Thr or Val. A substitution at sequence position 100may for example be a substitution Tyr 100→Ala, Arg, Glu, Gln, Gly, Serand Val. A substitution at sequence position 106 may for example be asubstitution Tyr 106→Ile, Gly, Phe, Val or Arg. A mutein of theinvention may in some embodiments have the set of amino acidsubstitutions, relative to the linear polypeptide sequence of a maturehuman Lipocalin 2, of Asn 96→Val, and Tyr 100→Gln. In such an embodimentthe tyrosine at position 106 may be unchanged. A mutein of the inventionmay in some embodiments have the set of amino acid substitutions,relative to the linear polypeptide sequence of a mature human Lipocalin2, of Asn 96→Arg, Tyr 100→Glu, and Tyr 106→Phe. In some embodiments amutein of the invention may have the set of amino acid substitutions ofAsn 96→Asp, Tyr 100→Ser and Tyr 106→Gly. A mutein of the invention mayin some embodiments have the set of amino acid substitutions of Asn96→Gly, Tyr 100→Gly and Tyr 106→Gly. A mutein of the invention may insome embodiments have the set of amino acid substitutions of Asn 96→Lys,Tyr 100→Ala and Tyr 106→Ile. In some embodiments a mutein of theinvention may have the set of amino acid substitutions of Asn 96→Ser,Tyr 100→Arg and Tyr 106→Val. A mutein of the invention may in someembodiments have the set of amino acid substitutions of Asn 96→Ser, Tyr100→Val and Tyr 106→Arg. In some embodiments a mutein of the inventionmay have the set of amino acid substitutions of Asn 96→Thr, Tyr 100→Valand Tyr 106→Gly. In some embodiments a mutein of the present inventionfurther includes a mutated amino acid residue at position 134 within thelinear polypeptide sequence of the mature human Lipocalin 2 In oneembodiment this substitution is Lys 134→Trp.

In some embodiments, a mutein of the present invention includes,typically in addition to a mutation at one or more of sequence positions96, 100 and 106 (supra), a mutated amino acid residue at any one or moreof the sequence positions corresponding to the sequence positions 52,68, 81, 127 of the linear polypeptide sequence of the mature humanLipocalin 2. The mutein may, for instance, include within the linearpolypeptide sequence of the mature human Lipocalin 2, a substitution Tyr52→His, Leu, Phe or Trp. The mutein may also include within the linearpolypeptide sequence of the mature human Lipocalin 2 a substitution Ser68→Arg. Gly or Ile. The mutein may also include a substitution Arg81→Glu, Gly or Gln. The mutein may, for instance, include within thelinear polypeptide sequence of the mature human Lipocalin 2 asubstitution Ser 127→Thr or Trp. A mutein of the invention may in someembodiments have the set of amino acid substitutions, relative to thelinear polypeptide sequence of a mature human Lipocalin 2, of Tyr52→His, Ser 68→Arg, Arg 81→Ser and Ser 127→Trp. In some embodiments amutein of the invention may have the set of amino acid substitutions ofTyr 52→Leu, Ser 68→, Arg 81→Glu and Ser 127→Trp. A mutein of theinvention may in some embodiments have the set of amino acidsubstitutions of Tyr 52→Phe, Ser 68→Gly, Arg 81→Gly and Ser 127→Trp. Amutein of the invention may in some embodiments have the set of aminoacid substitutions of Tyr 52→Trp. Ser 68→Ile, Arg 81→Gln and Ser127→Trp. In some embodiments a mutein of the invention may have the setof amino acid substitutions of Tyr 52→Trp. Ser 68→Arg, Arg 81→Glu andSer 127→Trp. A mutein of the invention may in some embodiments have theset of amino acid substitutions, in relation to the sequence of a maturehuman Lipocalin 2, of Tyr 52→Trp, Ser 68→Arg, Arg 81→Glu and Ser127→Thr. In some embodiments a mutein of the invention may have the setof amino acid substitutions of Tyr 52→Trp, Ser 68→Arg, Arg 81→Glu andSer 127→Trp.

In a further embodiment of the invention, the mutein includes at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20mutated amino acid residues at any of the sequence positionscorresponding to the sequence positions 33, 36, 40, 41, 42, 43, 44, 46,47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79,80, 81, 86, 87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132,134, 136 and 138 of the linear polypeptide sequence of hNGAL or thecorresponding sites on other lipocalins. In a further embodiment, themutein includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 mutated amino acid residues at any one of thesequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51,52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98,96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 ofthe linear polypeptide sequence of hNGAL or the corresponding sites onother lipocalins. In still a further embodiment, the mutein includes 18,19 or 20 mutated amino acid residues at any one of the sequencepositions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54,55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99,100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of thelinear polypeptide sequence of human Lipocalin 2 or the correspondingsites on other lipocalins.

A mutein of the invention may, for example, with respect to the maturehLcn2 wild type amino acid sequence, include one or more amino acidreplacements such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 of the group. Leu36→Ala, Cys, Thr, Val; Ala 40→Arg,Glu, Gly and Ser; Ile 41→Ile, Leu, Met or Val; Gln 49→Leu or Met; Leu70→Asp, Asn, Gln, Met or Phe; Arg 72→Glu. Gly, Leu or Val; Lys 73→Ala,Arg, Glu, Gly. Leu. Thr or Tyr; Asp 77→Arg, Glu, Gly. Leu. Ser or Val;Trp 79→Gly, Leu, Ser, Tyr or Val; Leu 103→Ala, Arg, Gly or Trp; Tyr106→Gly, Ile, Phe or Val; Lys 125→Arg, Leu, Met, Phe, Thr, or Val; andTyr 132→Leu or Val. A mutein of the invention may, for instance, havethe set of amino acid combinations, in relation to the linearpolypeptide sequence of a mature human Lipocalin 2, of Ala 36, Ser 40,Leu 41, Met 49, Asn 70, Gly 72, Gly 73, Ser 77, Leu 79, Leu 125 and Val132. A mutein of the invention may, for example, have the set of aminoacid combinations, in relation to the sequence of a mature humanLipocalin 2, of Leu 36, Arg 40, Val 41, Gln 49, Asp 70, Arg 72, Thr 73,Leu 77, Ser 79, Thr 125 and Val 132. In some embodiments a mutein of theinvention may have the set of amino acid combinations of Leu 36, Glu 40,Ile 41, Leu 49, Gln 70, Gly 72, Glu 73, Gly 77, Gly 79, Phe 125 and Val132. A mutein of the invention may also have the set of amino acidcombinations of Leu 36, Glu 40, Ile 41, Met 49, Met 70, Leu 72, Ala 73,Glu 77, Leu 79, Val 125, Val 132 or the set of amino acid combinationsof Leu 36, Glu 40, Val 41, Met 49, Met 70, Leu 72, Ala 73, Glu 77, Leu79, Thr 125 and Val 132. In some embodiments a mutein of the inventionmay have the set of amino acid combinations of Leu 36, Glu 40, Val 41,Met 49, Met 70, Leu 72, Ala 73, Glu 77, Leu 79, Val 125 and Val 132 orthe set of amino acid combinations of Thr 36, Ser 40, Ile 41, Gln 49,Phe 70, Glu 72. Gly 73, Arg 77. Val 79, Val 125 and Leu 132. As afurther example, a mutein of the invention may have the set of aminoacid combinations of Val 36, Glu 40, Met 41, Leu 49, Met 70, Glu 72, Tyr73, Val 77, Leu 79. Arg 125 and Val 132. A mutein of the invention mayalso have the set of amino acid combinations of Val 36, Gly 40, Leu 41,Leu 49, Leu 70, Val 72, Arg 73, Arg 77, Tyr 79, Met 125 and Val 132.

In one embodiment of the present invention, the mutein includes mutatedamino acid residues at at least any 10, 14, 15, 20, 22, 24, 26, 28, 29,30, 31, 32, 33, 35 or all 45 of the above-listed sequence positions.

A mutein of the invention, which binds to Hepcidin, can include withrespect to the mature human Lipocalin 2 wild type amino acid sequence(Lcn2) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17amino acid replacements which include, but are not limited to, Leu36→Valor Cys; Ala40→Tyr or Lys or Val; Ile41→Thr or Ser or Leu; Gln49→Leu orTrp; Leu70→Gly; Arg72→Gly or Asp; Lys73→Leu or Thr or Asp; Asp77→Asn orHis or Leu; Trp79→Lys; Asn96→Ile or Arg; Tyr100→Gln or Arg or Glu;Leu103→Met or Arg or Gly; Tyr106→Tyr or Ala or Trp; Lys125→Thr or Val orGlu; Ser127→Gly or Gln or Ala; Tyr132→Met or Ser or Thr, and Lys134→Asn

In one embodiment, a mutein of the invention, which binds to Hepcidinincludes the following amino acid replacements: Leu36→Val; Ala40→Tyr;Ile41→Thr; Gln49→Leu; Leu70→Gly; Lys73→Leu; Asp77→Asn; Trp79→Lys;Asn96→Ile; Tyr100→Gln; Leu103→Met; Lys125→Thr; Ser127→Gly; Tyr132→Met;and Lys134→Asn. In a further embodiment, a mutein of the invention,which binds to Hepcidin, includes the following amino acid replacementsLeu36→Val; Ala40→Lys; Ile41→Ser; Gln49→Trp; Leu70→Gly; Arg72→Gly;Lys73→Thr, Asp77→His; Trp79→Lys; Asn96→Arg; Tyr100→Arg; Leu103→Arg;Tyr106→Ala; Lys125→Val; Ser127→Gln. Tyr132→Ser; and Lys134→Asn. Inanother embodiment, a mutein of the invention, which binds to Hepcidin,includes the following amino acid replacements Leu36→Cys; Ala40→Val;Ile41→Leu; Gln49→Leu; Leu70→Gly; Arg72→Asp; Lys73→Asp; Asp77→Leu;Trp79→Lys; Asn96→Arg; Tyr100→Glu; Leu103→Gly; Tyr106→Trp; Lys125→Glu;Ser127→Ala; Tyr132→Thr; and Lys134→Asn.

A mutein according to the present invention may further include, withrespect to the mature hLcn2 wild type amino acid sequence, the aminoacid replacement Gln 28→His. A mutein according to the invention mayalso include, relative to the mature hLcn2 wild type amino acidsequence, the amino acid replacement Lys 62→Arg. Further, a muteinaccording to the present invention may include, relative to the maturehLcn2 wild type amino acid sequence, the amino acid replacement Phe71→Pro or Ser. A further amino acid replacement that may be present in amutein of the present invention, relative to the mature hLcn2 wild typeamino acid sequence, is the replacement Lys 74→Glu. Yet a further aminoacid replacement that may be included in a mutein of the invention isthe replacement Lys 75→Glu. A mutein of the invention may also include,with respect to the mature hLcn2 wild type amino acid sequence, theamino acid replacement Cys 87→Ser. A mutein of the invention may alsoinclude the amino acid replacement Ser 146→Pro. A further amino acidreplacement that may be present in a mutein of the present invention,relative to the mature hLcn2 wild type amino acid sequence, is thereplacement Glu 147→Gly. A mutein of the invention may include furtheramino acid replacements. The muteins can further include amino acidreplacements, such as Tyr52→Gln or Val; Ser68→Lys or Asn; or Arg81→Trp,Asn or His.

A mutein of the invention typically exists as monomeric protein.However, it is also possible that a lipocalin mutein of the invention isable to spontaneously dimerise or oligomerise. Although the use oflipocalin muteins that form stable monomers may be preferred for someapplications, e.g. because of faster diffusion and better tissuepenetration, the use of lipocalin muteins that form stable homodimers ormultimers may be advantageous in other instances, since such multimerscan provide for a (further) increased affinity and/or avidity to a giventarget. Furthermore, oligomeric forms of the lipocalin mutein may haveslower dissociation rates or prolonged serum half-life.

It is also noted that the complex formation between the respectivemutein and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(D) (for example fluorescence titration, competition ELISA orsurface plasmon resonance, just to name a few) or even the mathematicalalgorithm which is used for evaluation of the experimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivemutein and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means that there may be a slight deviation in the measuredK_(D) values or a tolerance range depending, for example, on whether theKO value was determined by surface plasmon resonance (Biacore), bycompetition ELISA, or by “direct ELISA.”

In one embodiment, the muteins disclosed herein can be linked, either N-or C-terminal to an affinity tag such as pentahistidine tag, ahexahistidine tag or a steptavidin tag (e.g. Streptag®). Thus, thepresent application encompasses also all explicitly and genericdescribed muteins equipped with such tags.

The term “fragment” as used in the present invention in connection withthe feature lipocalin mutein fragment relates to proteins or peptidesderived from full-length mature Lcn 2 that are N-terminally and/orC-terminally shortened, i.e. lacking at least one of the N-terminaland/or C-terminal amino acids. Such fragments include preferably atleast 10, more preferably 20, most preferably 30 or more consecutiveamino acids of the primary sequence of mature Lcn 2 and are usuallydetectable in an immunoassay of mature Lcn 2. The word “detect” or“detecting” as used herein is understood both on a quantitative and aqualitative level, as well as a combination thereof. It thus includesquantitative, semi-quantitative and qualitative measurements of amolecule of interest. Accordingly, the presence or absence of a moleculesuch as a hepcidin, e.g. in a sample, as well as its concentration orlevel may be determined.

Also included in the scope of the present invention are the abovemuteins, which have been altered with respect to their immunogenicity,to reduce any detected immunogenicity by employing methods known to theskilled worker in the field.

Cytotoxic T-cells recognize peptide antigens on the cell surface of anantigen-presenting cell in association with a class I majorhistocompatibility complex (MHC) molecule. The ability of the peptidesto bind to MHC molecules is allele specific and correlates with theirimmunogenicity. To reduce the immunogenicity of a given protein, theability to predict which peptides in a protein have the potential tobind to a given MHC molecule is of great value. Approaches that employ acomputational threading approach to identify potential T-cell epitopeshave been previously described to predict the binding of a given peptidesequence to MHC class I molecules (Altuvia et al. (1995) J. Mol. Biol.249: 244-250). Such an approach may also be utilized to identifypotential T-cell epitopes in the muteins of the invention and to make,depending on its intended use, a selection of a specific mutein on thebasis of its predicted immunogenicity. It may be furthermore possible tosubject peptide regions that have been predicted to contain T-cellepitopes to additional mutagenesis to reduce or eliminate these T-cellepitopes and thus minimize immunogenicity. The removal of amphipathicepitopes from genetically engineered antibodies has been described(Mateo et al. (2000) Hybridoma 19(6):463-471) and may be adapted to themuteins of the present invention. The muteins thus obtained may possessa minimized immunogenicity, which is desirable for their use intherapeutic and diagnostic applications, such as those described below.

For some applications, it is also useful to employ the muteins of theinvention in a conjugated form. Accordingly, the invention is alsodirected to lipocalin muteins which are conjugated to a compound whichcan include, but is not limited to organic molecules, an enzyme label, acolored label, a cytostatic agent, a toxin, a label that can bephotoactivated and which is suitable for use in photodynamic therapy, afluorescent label, a radioactive label, a chromogenic label, aluminescent label, metal complexes, metal, such as colloidal gold,haptens, digoxigenin, biotin, a chemotherapeutic metal, or achemotherapeutic metal, to name only a few evocative examples. Themutein may also be conjugated to an organic drug molecule. Theconjugation can be carried out using any conventional coupling methodknown in the art.

The term “organic molecule” or “small organic molecule” as used hereinfor the non-natural target denotes an organic molecule comprising atleast two carbon atoms, but preferably not more than 7 or 12 rotatablecarbon bonds, having a molecular weight in the range between 100 and2000 Dalton, preferably between 100 and 1000 Dalton, and optionallyincluding one or two metal atoms.

In general, it is possible to label a lipocalin mutein described hereinwith any appropriate chemical substance or enzyme, which directly orindirectly generates a detectable compound or signal in a chemical,physical, optical, or enzymatic reaction. An example for a physicalreaction and at the same time optical reaction/marker is the emission offluorescence upon irradiation. Alkaline phosphatase, horseradishperoxidase or β-galactosidase are examples of enzyme labels (and at thesame time optical labels) which catalyze the formation of chromogenicreaction products. In general, all labels commonly used for antibodies(except those exclusively used with the sugar moiety in the Fc part ofimmunoglobulins) can also be used for conjugation to the muteins of thepresent invention. The muteins of the invention may also be conjugatedwith any suitable therapeutically active agent, e.g., for the targeteddelivery of such agents to a given cell, tissue or organ or for theselective targeting of cells, e.g., of tumor cells without affecting thesurrounding normal cells. Examples of such therapeutically active agentsinclude radionuclides, toxins, small organic molecules, and therapeuticpeptides (such as peptides acting as agonists/antagonists of a cellsurface receptor or peptides competing for a protein binding site on agiven cellular target). Examples of suitable toxins include, but are notlimited to pertussis-toxin, diphtheria toxin, ricin, saporin,pseudomonas exotoxin, calicheamicin or a derivative thereof, a taxoid, amaytansinoid, a tubulysin or a dolastatin analogue. The dolastatinanalogue may be auristatin E, monomethylauristatin E, auristatin PYE andauristatin PHE. Examples of cytostatic agent include, but are notlimited to Cisplatin, Carboplatin, Oxaliplatin, 5-Fluorouracil. Taxotere(Docetaxel), Paclitaxel, Anthracycline (Doxorubicin), Methotrexate,Vinblastin, Vincristine. Vindesine, Vinorelbine, Dacarbazine,Cyclophosphamide, Etoposide. Adriamycine, Camptotecine. CombretatastinA-4 related compounds, sulfonamides, oxadiazolines,benzo[b]thiophenessynthetic spiroketal pyrans, monotetrahydrofurancompounds, curacin and curacin derivatives, methoxyestradiol derivativesand Leucovorin. The lipocalin muteins of the invention may also beconjugated with therapeutically active nucleic acids such as antisensenucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes.Such conjugates can be produced by methods well known in the art.

In one embodiment, the muteins of the invention may also be coupled to atargeting moiety that targets a specific body region in order to deliverthe inventive muteins to a desired region or area within the body. Oneexample wherein such modification may be desirable is the crossing ofthe blood-brain-barrier. In order to cross the blood-brain barrier, themuteins of the invention may be coupled to moieties that facilitate theactive transport across this barrier (see Gaillard P J, et al. (2005)International Congress Series. 1277.185-198 or Gaillard P J, et al.(2005) Expert Opin Drug Deliv. 2(2), 299-309). Such compounds are forexample available under the trade name 28-Trans™ (to-BBB technologiesBV, Leiden, NL). Other exemplary targeting molecules to which themuteins of the present invention may be coupled include antibodies,antibody fragments or lipocalin muteins with affinity for a desiredtarget molecule. The target molecule of the targeting moieties may, forexample, be a cell-surface antigen. Cell-surface antigens may bespecific for a cell or tissue type, such as, for example, cancer cells.Illustrative examples of such cell surface proteins are HER-2 orproteoglycans such as NEU-2.

As indicated above, a mutein of the invention may in some embodiments beconjugated to a compound that extends the serum half-life of the mutein(in this regard see also PCT publication WO 2006/56464 where suchconjugation strategies are described with references to muteins of humanneutrophil gelatinase-associated lipocalin with binding affinity forCTLA-4). The compound that extends the serum half-life may be apolyalkylene glycol molecule, such as polyethylene (PEG) or an activatedderivative thereof; hydroxyethyl starch, fatty acid molecules, such aspalmitic acid (Vajo & Duckworth (2000) Pharmacol. Rev. 52, 1-9), an Fcpart of an immunoglobulin, a C_(H)3 domain of an immunoglobulin, aC_(H)4 domain of an immunoglobulin, albumin or a fragment thereof, analbumin binding peptide, an albumin binding protein, transferrin, or thetag Pro-Ala-Ser, to name only a few. The albumin binding protein may bea bacterial albumin binding protein, an antibody, an antibody fragmentincluding domain antibodies (see U.S. Pat. No. 6,696,245, for example),or a lipocalin mutein with binding activity for albumin. Accordingly,suitable conjugation compounds for extending the half-life of alipocalin mutein of the invention include albumin (Osborn et al. (2002)J. Pharmacol. Exp. Ther. 303, 540-548), or an albumin binding protein,for example a bacterial albumin binding domain, such as the one ofstreptococcal protein G (König, T. and Skerra, A. (1998) J. Immunol.Methods 218, 73-83). Other examples of albumin binding peptides that canbe used as conjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln. His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

In other embodiments, albumin itself or a biological active fragment ofalbumin can be used as compound of a lipocalin mutein of the inventionthat extends the serum half-life of the mutein. The term “albumin”includes all mammal albumins such as human serum albumin or bovine serumalbumin or rat albumin. The albumin or fragment thereof can berecombinantly produced as described in U.S. Pat. No. 5,728,553 orEuropean patent applications EP 0 330 451 and EP 0 361 991. Recombinanthuman albumin (Recombumin®) for use as a protein stabilizer is forexample available from Novozymes Delta Ltd. (Nottingham, UK).

If the albumin-binding protein is an antibody fragment it may be adomain antibody Domain Antibodies (dAbs) are engineered to allow precisecontrol over biophysical properties and in vivo half-life to create theoptimal safety and efficacy product profile. Domain Antibodies are forexample commercially available from Domantis Ltd. (Cambridge, UK and MA.USA).

Using transferrin as a moiety to extend the serum half-life of themuteins of the invention, the muteins can be genetically fused to the Nor C terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation. PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer is also commercially available fromNovozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the muteins of the invention, the SynFusion™technology, commercially available from Syntonix Pharmaceuticals, Inc(MA, USA), may be used. The use of this Fc-fusion technology allows thecreation of longer-acting biopharmaceuticals and may for examplecomprise two copies of the mutein linked to the Fc region of an antibodyto improve pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of a mutein of theinvention is to fuse the N- or C-terminus of a mutein of the inventionto long, unstructured, flexible glycine-rich sequences (for examplepoly-glycine with about 20 to 80 consecutive glycine residues). Thisapproach disclosed in WO2007/038619, for example, has also been term“rPEG” (recombinant PEG).

If polyalkylene glycol is used as compound that extends the half-life ofthe mutein, the polyalkylene glycol can be substituted or unsubstituted.It can also be an activated polyalkylene derivative. Examples ofsuitable compounds are polyethylene glycol (PEG) molecules as describedin WO 99/84016, in U.S. Pat. No. 6,177,074 or in U.S. Pat. No. 6,403,564in relation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (see for example. Fuertges et al. (1990) “TheClinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins” J.Control. Release 11, 139-148). The molecular weight of such a polymer,preferrably polyethylene glycol, may range from about 300 to about70.000 Dalton, including for example, polyethylene glycol with amolecular weight of about 10 000, of about 20.000, of about 30.000 or ofabout 40.000 Dalton. Moreover. e.g. as described in U.S. Pat. Nos.6,500,930 or 6,620,413, carbohydrate oligo- and polymers such as starchor hydroxyethyl starch (HES) can be conjugated to a mutein of theinvention for the purpose of serum half-life extension.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above compounds to the muteins of theinvention artificial amino acids may be introduced by mutagenesis.Generally, such artificial amino acids are designed to be more reactiveand thus to facilitate the conjugation to the desired moiety. Oneexample of such an artificial amino acid that may be introduced via anartificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins disclosed herein, it may beadvantageous to use them in the form of fusion proteins. In someembodiments, the inventive mutein is fused at its N-terminus and/or it'sC-terminus to a protein, a protein domain or a peptide such as a signalsequence and/or an affinity tag.

For pharmaceutical applications, a mutein of the invention may be fusedto a fusion partner that extends the in vivo serum half-life of themutein (see again PCT publication WO 2006/56464 where suitable fusionpartner are described with references to muteins of human neutrophilegelatinase-associated lipocalin with binding affinity for CTLA-4).Similar to the conjugated compounds described above, the fusion partnermay be an Fc part of an immunoglobulin, a C_(H)3 domain of animmunoglobulin, a C_(H)4 domain of an immunogloubulin, albumin, analbumin binding peptide or an albumin binding protein, to name only afew. Again, the albumin binding protein may be a bacterial albuminbinding protein or a lipocalin mutein with binding activity for albumin.Accordingly, suitable fusion partners for extending the half-life of alipocalin mutein of the invention include albumin (Osbom, B. L. et al.(2002) supra J. Pharmacol. Exp. Ther. 303, 540-548), or an albuminbinding protein, for example, a bacterial albumin binding domain, suchas streptococcal protein G (König, T, and Skerra, A. (1998) supra J.Immunol. Methods 218, 73-83). The albumin binding peptides described inDennis et al. supra (2002) or US patent application 2003/0069395 havinga Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp.Asn. Set, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr, and Xaa₄ is Asp, Gly, Leu. Phe, Ser, or Thrcan also be used as fusion partner. It is also possible to use albuminitself or a biological active fragment of albumin as fusion partner of alipocalin mutein of the invention. The term “albumin” includes allmammal albumins such as human serum albumin or bovine serum albumin orrat serum albumin. The recombinant production of albumin or fragmentsthereof is well known in the art and for example described in U.S. Pat.No. 5,728,553, European patent application EP 0 330 451 or EP 0 361 991

The fusion partner may confer new characteristics to the inventivelipocalin mutein such as enzymatic activity or binding affinity forother molecules. Examples of suitable fusion proteins are alkalinephosphatase, horseradish peroxidase, gluthation-S-transferase, thealbumin-binding domain of protein G, protein A, antibody fragments,oligomerization domains, lipocalin muteins of same or different bindingspecificity (which results in the formation of “duocalins,” cf.Schlehuber, S., and Skerra, A. (2001), Duocalins, engineeredligand-binding proteins with dual specificity derived from the lipocalinfold (Biol. Chem. 382, 1335-1342), or toxins.

In particular, it may be possible to fuse a lipocalin mutein of theinvention with a separate enzyme active site such that both “components”of the resulting fusion protein together act on a given therapeutictarget. The binding domain of the lipocalin mutein attaches to thedisease-causing target, allowing the enzyme domain to abolish thebiological function of the target.

Affinity tags such as the Strep-Tag® or Strep-Tag® II (Schmidt. T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of preferred fusion partners. Finally, proteinswith chromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for a lipocalin mutein of the invention as well.

The term “fusion protein” as used herein also includes lipocalin muteinsaccording to the invention containing a signal sequence. Signalsequences at the N-terminus of a polypeptide direct this polypeptide toa specific cellular compartment, for example the periplasm of E. coli orthe endoplasmatic reticulum of eukaryotic cells. A large number ofsignal sequences is known in the art. A preferred signal sequence forsecretion a polypeptide into the periplasm of E. coli is the OmpA-signalsequence.

The present invention also relates to nucleic acid molecules (DNA andRNA) comprising nucleotide sequences coding for muteins as describedherein. Since the degeneracy of the genetic code permits substitutionsof certain codons by other codons specifying the same amino acid, theinvention is not limited to a specific nucleic acid molecule encoding amutein of the invention but includes all nucleic acid moleculescomprising nucleotide sequences encoding a functional mutein.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it includes sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter which, inprokaryotes, contains both the promoter per so, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the invention can include aregulatory sequence, preferably a promoter sequence. In anotherpreferred embodiment, a nucleic acid molecule of the invention includesa promoter sequence and a transcriptional termination sequence. Suitableprokaryotic promoters are, for example, the tet promoter, the lacUV5promoter or the T7 promoter. Examples of promoters useful for expressionin eukaryotic cells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the invention can also be part of a vectoror any other kind of cloning vehicle, such as a plasmid, a phagemid, aphage, a baculovirus, a cosmid or an artificial chromosome.

The DNA molecule encoding lipocalin muteins of the invention, and inparticular a cloning vector containing the coding sequence of such alipocalin mutein can be transformed into a host cell capable ofexpressing the gene. Transformation can be performed using standardtechniques (Sambrook, J et al. (1989), supra).

Thus, the invention is also directed to a host cell containing a nucleicacid molecule as disclosed herein.

The invention also relates to a method for the production of a mutein ofthe invention, wherein the mutein, a fragment of the mutein or a fusionprotein of the mutein and another polypeptide is produced starting fromthe nucleic acid coding for the mutein by means of genetic engineeringmethods. The method can be carried out in vivo, the mutein can forexample be produced in a bacterial or eucaryotic host organism and thenenriched, purified or isolated from this host organism or its culture.It is also possible to produce a protein in vitro, for example by use ofan in vitro translation system. The term “enriched” means that themutein or a functional fragment thereof constitutes a significantlyhigher fraction of the total protein present in a sample or solution ofinterest than in a sample or solution from which it was taken.Enrichment may for instance include the isolation of a certain fractionfrom a cell extract. This may be obtained by standard techniques such ascentrifugation. Examples of other means of enrichment are filtration ordialysis, which may for instance be directed at the removal of undesiredmolecules below a certain molecular weight or a precipitation usingorganic solvents or ammonium sulphate. Purification may for instanceinclude a chromatographic technique, for example gel filtration, ionexchange chromatography, affinity purification, hydrophobic interactionchromatography or hydrophobic charge induction chromatography. Anotherexample for a purification is an electrophoretic technique, such aspreparative capillary electrophoresis. Isolation may include thecombination of similar methods. As used herein, “substantially pure” or“substantially purified” means a compound or species that is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In someembodiments, a substantially purified composition is a composition inwhich the species includes at least about 50 percent (on a molar basis)of all molecular or, as applicable, all macromolecular species present.In certain embodiments, a substantially pure composition will have morethan about 80%, about 85%, about 90%, about 95%, or about 99% of allmolecular or, as applicable, all macromolar species present in thecomposition.

When producing the mutein in vivo, a nucleic acid encoding a mutein ofthe invention is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector comprising a nucleic acid molecule encoding a mutein ofthe invention using established standard methods (Sambrook, J. et al.(1989), supra). The host cell is then cultured under conditions, whichallow expression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide. Subsequently, the polypeptide is recoveredeither from the cell or from the cultivation medium.

In one aspect, the present invention relates to a method for thegeneration of a mutein which binds hepcidin, comprising:

subjecting a nucleic acid molecule encoding a lipocalin to mutagenesis,resulting in one or more mutein nucleic acid molecule(s).

The method can further include:

expressing the one more mutein nucleic acid molecule(s) obtained in (a)in a suitable expression system,

bringing the plurality of muteins into contact with at least a fragmentor a mature form of hepcidin, and

enriching the one or more mutein(s) having a detectable binding affinityfor a given target by means of selection and/or isolation.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the lipocalin, including Lcn 2 (hNGAL,Swiss-Prot data bank entry P80188) can be substituted by at least oneamino acid that is not present at this specific position in therespective natural polypeptide sequence. The term “mutagenesis” alsoincludes the (additional) modification of the length of sequencesegments by deletion or insertion of one or more amino acids. Thus, itis within the scope of the invention that, for example, one amino acidat a chosen sequence position is replaced by a stretch of three randommutations, leading to an insertion of two amino acid residues comparedto the length of the respective segment of the wild type protein. Suchan insertion of deletion may be introduced independently from each otherin any of the peptide segments that can be subjected to mutagenesis inthe invention. In one exemplary embodiment of the invention, aninsertion of several mutations may be introduced into the loop AB of thechosen lipocalin scaffold (cf. International Patent Application WO2005/019256 which is incorporated by reference its entirety herein). Theterm “random mutagenesis” means that no predetermined single amino acid(mutation) is present at a certain sequence position but that at leasttwo amino acids can be incorporated with a certain probability at apredefined sequence position during mutagenesis.

In one non-limiting approach, the coding sequence of human Lipocalin 2can be used as a starting point for the mutagenesis of the peptidesegments selected in the present invention. For the mutagenesis of therecited amino acid positions, the person skilled in the art has at hisdisposal the various established standard methods for site-directedmutagenesis (Sambrook, J. et al. (1989), supra). A commonly usedtechnique is the introduction of mutations by means of PCR (polymerasechain reaction) using mixtures of synthetic oligonucleotides, which beara degenerate base composition at the desired sequence positions. Othersimilar techniques are well known to those of skill in the art.

The nucleic acid molecules defined above can be connected by ligationwith the missing 5′- and 3′-sequences of a nucleic acid encoding alipocalin polypeptide and/or the vector, and can be cloned in a knownhost organism. A multitude of established procedures are available forligation and cloning (Sambrook, J. et al. (1989), supra). For example,recognition sequences for restriction endonucleases also present in thesequence of the cloning vector can be engineered into the sequence ofthe synthetic oligonucleotides. Thus, after amplification of therespective PCR product and enzymatic cleavage the resulting fragment canbe easily cloned using the corresponding recognition sequences.

Longer sequence segments within the gene coding for the protein selectedfor mutagenesis can also be subjected to random mutagenesis via knownmethods, for example by use of the polymerase chain reaction underconditions of increased error rate, by chemical mutagenesis or by usingbacterial mutator strains. Such methods can also be used for furtheroptimization of the target affinity or specificity of a lipocalin muteinMutations possibly occurring outside the segments of experimentalmutagenesis are often tolerated or can even prove to be advantageous,for example if they contribute to an improved folding efficiency orfolding stability of the lipocalin mutein.

In a further embodiment, the method includes subjecting the nucleic acidmolecule to mutagenesis at nucleotide triplets coding for at least any1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20of the sequence positions corresponding to the sequence positions 33,36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68,70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103, 106,107, 110, 111, 125, 127, 132, 134, 136 and/or 138 of the linearpolypeptide sequence of the lipocalin, or, for example, human Lipocalin2. Such a nucleic acid may subjected to mutagenesis and introduced intoa suitable bacterial or eukaryotic host organism by using recombinantDNA technology. Obtaining a nucleic acid library of a lipocalin can becarried out using any suitable technique that is known in the art forgenerating lipocalin muteins with antibody-like properties, i.e. muteinsthat have affinity towards a given target. Examples of suchcombinatorial methods are described in detail in the internationalpatent applications WO 99/16873, WO 00/75308, WO 03/029471, WO03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255, WO 2005/019256,or WO 2006/56464 for instance. The content of each of these patentapplications is incorporated by reference herein in their entirety.After expression of the nucleic acid sequences that were subjected tomutagenesis in an appropriate host, the clones carrying the geneticinformation for the plurality of respective lipocalin muteins, whichbind a given target can be selected from the library obtained. Wellknown techniques can be employed for the selection of these clones, suchas phage display (reviewed in Kay, B. K. et al (1996) supra; Lowman, H.B (1997) supra or Rodi, D. J., and Makowski, L. (1999) supra), colonyscreening (reviewed in Pini. A. et al. (2002) Comb. Chem. HighThroughput Screen. 5, 503-510), ribosome display (reviewed in Amstutz,P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405) or mRNA display asreported in Wilson, D. S. et al. (2001) Proc. Natl. Acad. Sci. USA 98,3750-3755 or the methods specifically described in WO 99/16873, WO00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO2005/019255, WO 20051019256, or WO 2006/56464.

In accordance with this disclosure, another embodiment of the abovemethods comprises:

(i) providing at least a fragment of hepcidin as a given target/ligandfor example,

contacting the plurality of muteins with said target/ligand in order toallow formation of complexes between said ligand and muteins havingbinding affinity for said target/ligand, and

removing muteins having no or no substantial binding affinity.

In one embodiment of the methods of the invention, the selection bindingaffinity is carried out under competitive conditions. Competitiveconditions as used herein means that selection of muteins encompasses atleast one step in which the muteins and the fragment of hepcidin or amature hepcidin such as Hepcidin-25 (target) are brought in contact inthe presence of an additional ligand, which competes with binding of themuteins to the target (hepcidin). This additional target may be anotherform of hepcidin, for example Hepcidin-20 (in case muteins are to beselected that selectively bind Hepcidin-25 or even the five N-terminalresidues of Hepcidin-25 (as indicated earlier, it is presently assumedthat the iron-regulating bioactivity is almost exclusively due to the 25amino acid form Hepcidin-25, indicating that the five N-terminal aminoacids are essential for this activity, Kenma et al., supra), an excessof the target itself or any other non-physiological ligand of thehepcidin that binds at least an overlapping epitope to the epitoperecognized by the muteins of the invention and thus interferes withtarget (hepcidin) binding of the muteins. Alternatively, the additionalligand competes with binding of the muteins by complexing an epitopedistinct from the binding site of the muteins to the target byallosteric effects. Accordingly, any fragment, precursor or mature formof Hepcidin can be used in the generation of muteins of the invention.

A further embodiment of the methods of the invention involves operablyfusing a nucleic acid coding for the plurality of muteins of of theinvention and resulting from mutagenesis at the 3′ end with a genecoding for the coat protein pill of a filamentous bacteriophage of theM13-family or for a fragment of this coat protein, in order to select atleast one mutein for the binding of a given ligand.

The fusion protein may include additional components such as an affinitytag, which allows the immobilization, detection and/or purification ofthe fusion protein or its parts. Furthermore, a stop codon can belocated between the sequence regions encoding the lipocalin or itsmuteins and the phage capsid gene or fragments thereof, wherein the stopcodon, preferably an amber stop codon, is at least partially translatedinto an amino acid during translation in a suitable suppressor strain.

For example, the phasmid vector pTLPC27, now also called pTlc27 that isdescribed here can be used for the preparation of a phagemid libraryencoding muteins of the invention. The inventive nucleic acid moleculescoding for muteins of the invention can be inserted into the vectorusing the two BstXI restriction sites. After ligation a suitable hoststrain such as E. coli XL1-Blue is transformed with the resultingnucleic acid mixture to yield a large number of independent clones. Arespective vector can be generated for the preparation of ahyperphagemid library, if desired.

Once a mutein with affinity to a given target has been selected, it isadditionally possible to subject such a mutein to another mutagenesis inorder to subsequently select variants of even higher affinity orvariants with improved properties such as higher thermostability,improved serum stability, thermodynamic stability, improved solubility,improved monomeric behavior, improved resistance against thermaldenaturation, chemical denaturation, proteolysis, or detergents etc.This further mutagenesis, which in case of aiming at higher affinity canbe considered as in vitro “affinity maturation,” can be achieved by sitespecific mutation based on rational design or a random mutation. Anotherpossible approach for obtaining a higher affinity or improved propertiesis the use of error-prone PCR, which results in point mutations over aselected range of sequence positions of the lipocalin mutein. Theerror-prone PCR can be carried out in accordance with any known protocolsuch as the one described by Zaccolo at al. (1996) J. Mol. Biol. 255,589-603. Other methods of random mutagenesis that are suitable for suchpurposes include random insertion/deletion (RID) mutagenesis asdescribed by Murakami et al. (2002) Nat. Biotechnol. 20, 76-81 ornonhomologous random recombination (NRR) as described by Bittker et al.(2002) Nat. Biotechnol. 20, 1024-1029. If desired, affinity maturationcan also be carried out according to the procedure described in WO00/75308 or Schlehuber et al. (2000) J. Mol. Biol. 297, 1105-1120, wheremuteins of the bilin-binding protein having high affinity to digoxigeninwere obtained. A further approach for improving the affinity is to carryout positional saturation mutagenesis. In this approach “small” nucleicacid libraries can be created in which amino acid exchanges/mutationsare only introduced at single positions within any of the four loopsegments. These libraries are then directly subjected to a selectionstep (affinity screening) without further rounds of panning. Thisapproach allows the identification of residues that contribute toimproved binding of the desired target and allows identification of “hotspots” that are important for the binding.

In one embodiment, the above method for modifying a mutein furtherincludes introducing a Cys residue at at least one of any of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of humanLipocalin 2 and coupling a moiety that is able to modify the serum halftime of said mutein via the thiol group of a Cys residue introduced atat least one of any of the sequence positions that correspond tosequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 ofthe wild type sequence of hNGAL. The moiety that is able to modify theserum half time of said mutein may be selected from the group consistingof a polyalkylene glycol molecule and hydroxyethylstarch.

Where a protein of the invention is a human Lipocalin 2 mutein of theinvention, the naturally occurring disulfide bond between Cys 76 and Cys175 may be removed. Accordingly, such muteins (or any other humanLipocalin 2 mutein that does not include an intramolecular disulfidebond) can be produced in a cell compartment having a reducing redoxmilieu, for example, in the cytoplasma of Gram-negative bacteria.

In case a lipocalin mutein of the invention includes intramoleculardisulfide bonds, it may be preferred to direct the nascent polypeptideto a cell compartment having an oxidizing redox milieu using anappropriate signal sequence. Such an oxidizing environment may beprovided by the periplasm of Gram-negative bacteria such as E. coli, inthe extracellular milieu of Gram-positive bacteria or in the lumen ofthe endoplasmatic reticulum of eukaryotic cells and usually favors theformation of structural disulfide bonds.

It is, however, also possible to produce a mutein of the invention inthe cytosol of a host cell, preferably E. coli. In this case, thepolypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi et al. (2002) J. Mol. Biol. 315,1-8).

However, a mutein of the invention may not necessarily be generated orproduced only by use of genetic engineering. Rather, a lipocalin muteincan also be obtained by chemical synthesis such as Merrifield solidphase polypeptide synthesis or by in vitro transcription andtranslation. It is for example possible that promising mutations areidentified using molecular modeling and then to synthesize the wanted(designed) polypeptide in vitro and investigate the binding activity fora given target. Methods for the solid phase and/or solution phasesynthesis of proteins are well known in the art (reviewed, e.g., inLLoyd-Williams et al. (1997) Chemical Approaches to the Synthesis ofPeptides and Proteins. CRC Press, Boca Raton, Fields, GB, and Colowick(1997) Solid-Phase Peptide Synthesis. Academic Press, San Diego, orBruckdorfer et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

In another embodiment, the muteins of the invention may be produced byin vitro transcription/translation employing well-established methodsknown to those skilled in the art.

The invention also relates to a pharmaceutical composition that includesat least one inventive mutein referred to in the claims or a fusionprotein or conjugates thereof and, optionally, a pharmaceuticallyacceptable excipient.

The lipocalin muteins according to the invention can be administered viaany parenteral or non-parenteral (e.g. enteral) route that istherapeutically effective for proteinaceous drugs.

Accordingly, the muteins of the present invention can be formulated intocompositions using pharmaceutically acceptable ingredients as well asestablished methods of preparation (Gennaro and Gennaro (2000)Remington: The Science and Practice of Pharmacy, 20th Ed., LippincottWilliams & Wilkins. Philadelphia. Pa.). To prepare the pharmaceuticalcompositions, pharmaceutically inert inorganic or organic excipients canbe used.

A protein of the invention of the invention may also be used to target acompound to a pre-selected site. In one such embodiment, a protein ofthe invention is used for the targeting of a pharmaceutically activecompound to a pre-selected site in an organism or tissue, comprising:

a) conjugating the protein with said compound, and

b) delivering the protein/compound complex to the pre-selected site.

For such a purpose the mutein is contacted with the compound of interestin order to allow complex formation. Then the complex comprising themutein and the compound of interest are delivered to the pre-selectedsite. This may, for example, be achieved by coupling the mutein to atargeting moiety, such as an antibody, antibody fragment or lipocalinmutein or lipocalin mutein fragment with binding affinity for theselected target.

This use is in particular suitable, but not restricted to, fordelivering a drug (selectively) to a pre-selected site in an organism,such as an infected body part, tissue or organ which is supposed to betreated with the drug. Besides formation of a complex between mutein andcompound of interest, the mutein can also be reacted with the givencompound to yield a conjugate of mutein and compound. Similar to theabove complex, such a conjugate may be suitable to deliver the compoundto the pre-selected target site.

Such a conjugate of mutein and compound may also include a linker thatcovalently links mutein and compound to each other. Optionally, such alinker is stable in the bloodstream but is cleavable in a cellularenvironment.

The muteins disclosed herein and its derivatives can thus be used inmany fields similar to antibodies or fragments thereof. In addition totheir use for binding to a support, allowing the target of a givenmutein or a conjugate or a fusion protein of this target to beimmobilized or separated, the muteins can be used for labeling with anenzyme, an antibody, a radioactive substance or any other group havingbiochemical activity or defined binding characteristics. By doing so,their respective targets or conjugates or fusion proteins thereof can bedetected or brought in contact with them. For example, muteins of theinvention can serve to detect chemical structures by means ofestablished analytical methods (e.g. ELISA or Western Blot) or bymicroscopy or immunosensorics. Here, the detection signal can either begenerated directly by use of a suitable mutein conjugate or fusionprotein or indirectly by immunochemical detection of the bound muteinvia an antibody.

Numerous possible applications for the inventive muteins also exist inmedicine. In addition to their use in diagnostics and drug delivery, amutant polypeptide of the invention, which binds, for example, tissue-or tumor-specific cellular surface molecules can be generated. Such amutein may, for example, be employed in conjugated form or as a fusionprotein for “tumor imaging” or directly for cancer therapy.

In a further aspect, the present invention also encompasses the use of amutein according to the invention for the manufacture of apharmaceutical composition. The pharmaceutical composition thus obtainedmay be suited for reducing the level of a Hepcidin. The pharmaceuticalcomposition may be used as monotherapy or as combination therapy.Accordingly, the invention also relates to a mutein as defined above forthe treatment of a disease or disorder associated with an altered, e.g.increased or reduced level of a Hepcidin.

Diseases Associated with Hepcidin

Anemia is a disease associated with serum iron depletion leading to adecrease of hematological parameters such as red blood cell (RBC)counts, hematocrit (Ht), hemoglobin (Hb), serum iron level andtransferrin (Tf) saturation. This results in a decreased oxygen level inthe blood and is associated with a declined quality of life (QOL)described by weakness, poor concentration, shortness of breath anddyspnea. Severe anemia can lead to a fast heart rate, cardiacenlargement and heart failure. Anemia is often associated with chronickidney disease/established chronic kidney disease (CKD), anemia ofcancer (AC), chemotherapy induced anemia (CIA) and anemia of chronicdisease (ACD).

The effective management of anemia has a major impact on quality of lifeand may influence the survival of patients. The declined quality of lifecan be described by weakness, fatigue, poor concentration, shortness ofbreath up to dyspnea. Severe anemia is associated with a fast heartrate, and can lead to cardiac enlargements and heart failure.

The standard treatment of care are transfusions and the administrationof ESAs and iron. Nevertheless, new therapeutic approaches are desiredsince the standard treatments are associated with the followingdisadvantages or potential draw backs. Transfusion bears the risk ofhemolysis, infections and allergic reactions due to an incompatibleblood type. Iron treatment can lead to iron overload in long termtreatments and is not recommended for the treatment of anemia ofinflammation since iron contributes to inflammatory responses (e.g.inflammatory joint disease). As far as ESAs are concerned, about 40-50%of anemic patients are ESA non-responder with no or delayed Hb-responseonly after high dose ESA-treatment that are associated with safetyconcerns like poorer survival and shorter progression free survival timein cancer patients.

Iron deficiency anemia is a disorder of iron homeostasis that is easilycured by iron administration in contrast to anemia associated withinflammatory disease. Hepcidin is a parameter that allows distinguishingbetween these two disorders since the hepcidin level is only upregulatedin combination with inflammation.

Anemias associated with chronic inflammatory disease like chronicinfections, rheumatologic and systemic autoimmune disorders andinflammatory bowel disease are called anemia of inflammation (AI) oranemia of chronic disease (ACD). Hepcidin expression is induced by theinflammatory cytokine IL-6, as part of the inflammatory response,resulting in iron deficiency induced anemia and a blunted response toESAs.

Patients with established chronic kidney disease (chronic renal failure(CRF)) develop uremic anemia as one of the most obvious signs of thedisease. This symptom is caused by impeded renal production oferythropoietin (EPO). EPO controls red blood cell (RBC) production bypromoting survival, proliferation and differentiation of erythroidprogenitors in the bone marrow. Effective management of anemia inchronic renal failure (CRF) has a major impact on quality of life andmay influence survival. Supplementation with recombinant humanerythropoietin (rhEPO) is currently the standard treatment for anemia inthose patients. A response rate of 70-90% to various ESA's(erythropoesis stimulating agents) has been observed in clinical trialswith CRF patients. Only in patients with additional inflammatory diseasehepcidin plays a prominent role in the anemia associated with CKD.

Anemia is common in patients with cancer and has a multifactorialaetiology. It may be related to the malignancy itself and its extent, aswell as to the type, duration and intensity of myelosuppressivechemotherapy. Moreover, most patients with cancer have been shown tohave inappropriately low levels of circulating EPO for their degree ofanemia, reflecting a change in this homeostatic mechanism. The incidenceof anemia severe enough to result in blood transfusions may be as highas 60% in certain tumor types. Anemic patients with cancer mayexperience symptoms as fatigue, dizziness, shortness of breath, andcardiovascular symptoms such as palpitations and cardiac failure. Suchclinical sequelae may decrease the quality of life of these patients.Furthermore, a potential relationship between the correction of anemiaand increased survival in patients receiving chemotherapy has recentlybeen discussed. Currently, therapeutic options for anemia in cancerpatients are RBC transfusions or ESA's. Transfusion of RBCs can beassociated with non-hemolytic and hemolytic transfusion reactions, ironoverload in heavily transfused patients, or the transmission ofinfections. Safety and screening requirements in transfusion therapyhave increased the logistics and cost of transfusion therapy thusrestricting transfusions to cases of severe and/or symptomatic anemia.ESA's have provided an alternative to blood transfusions in thetreatment of symptomatic anemia which is still not severe enough tomerit transfusions with current policies. However, a clear dose responserelationship for ESA's has not been established, and 40% to 50% ofpatients show no Hb response at all or a delayed response. During thelast years important concerns have emerged regarding the impact of ESAson cancer patients' survival as well as their potential to increase therisk of thromboembolism (in march 2007 the FDA instituted a black-boxwarning about the possible association of ESAs with tumor promotion andthromboembolic events). There is raising evidence from the literaturethat ESA-resistance of cancer patients is not only predicted by amissing increase in Hb-response within 4 weeks of ESA administration butalso by an elevated hepcidin level—presumably as part of an inflammatoryresponse.

As explained above, Hepcidin is the central negative regulator of ironhomeostasis. Hepcidin production increases with iron loading andinflammation and decreases under low iron conditions and hypoxia.Hepcidin acts via binding to the only known mammalian cellular ironexporter, ferroportin, and induces its internalization and degradation.Since ferroportin is expressed in the duodenal enterocytes, spleen, andliver, hepcidin increase, and the subsequent decrease of ferroportin,results in the inhibition of duodenal iron absorption, release ofrecycled iron from macrophages, and mobilization of iron stores in theliver. Hepcidin is thought to play a critical role in the development ofanemia associated with inflammatory disease. Acute or chronicinflammatory conditions result in the up-regulation of hepcidinexpression, leading to iron deficiency, which can cause anemiaassociated with inflammatory disease (ACD), cancer (AC, CIA) and ChronicKidney Disease (CKD) (anemia of CKD).

A lipocalin mutein according to the invention may be used as anantagonist of a hepcidin (supra). In this regard a lipocalin muteinaccording to the invention, typically an isolated lipocalin mutein, maybe used in therapy, such as human therapy. A respective mutein iscapable of forming a complex with a hepcidin, e.g. a human hepcidin,typically with high affinity. Thereby the lipocalin mutein typicallyblocks the interaction with the hepcidin receptor ferroportin. As aresult internalization and degradation of ferroportin are prevented. Thelipocalin mutein thereby supports erythropoiesis by allowingmobilization of stored iron and improved enteral iron absorption. Anillustrative example of a subject in need of application of a respectiveantagonist of a hepcidin according to the invention is a subjecthyporesponsive to ESA-therapy (about 40-50% of patients) which isthought to be caused by the decreased availability of iron for thesynthesis of hemoglobin due to upregulated hepcidin. The term “subject”refers to a vertebrate animal, including a mammal, and in particular ahuman, in which case the term “patient” can also be used. In someembodiments, the subject may have a disorder that would benefit from adecreased level of a hepcidin such as hepcidin-25, a decrease inbioactivity of a hepcidin (e.g. hepcidin-25 bioactivity), and/or anincrease in serum iron level, reticulocyte count, red blood cell count,hemoglobin, and/or hematocrit.

A lipocalin mutein according to the invention may be used to increaseiron levels in a body fluid such as serum. It may also be used toincrease reticulocyte count, red blood cell count, hemoglobin, and/orhematocrit in a subject, e.g. a human. A pharmaceutical compositioncomprising a lipocalin mutein of the invention may be used in thisregard.

Another aspect of the present invention relates to a method of treatinga subject suffering from a disease or disorder that is associated withan altered level of a Hepcidin, such as an increased or a decreasedlevel of a Hepcidin. A respective disease or disorder may include agenetic or a non-genetic disease/disorder causing iron deficiency oroverload. A disease state or disorder may include an infectious diseaseinvolving e.g. bacteria, fungi, yeast or viruses. As explained above, insome embodiments the disease or disorder is anemia, including, but notlimited to, anemia resulting from infection, inflammation, chronicdisease, and/or cancer. It may in some embodiments include aninflammatory disease such as arthritis and certain cancer types, a liverdisease or a haematological disease. In some embodiments of the diseaseassociated with an increased level of a Hepcidin is an anemia or achronic kidney disease or an anemia associated with chronic kidneydisease. As already explained above, such a method involvesadministering a respective mutein of the invention or a pharmaceuticalcomposition comprising a mutein of the invention to a subject in needthereof.

A lipocalin mutein of the invention may for instance be used to treat asubject having an elevated level of hepcidin, a hepcidin-relateddisorder, a disorder of iron homeostasis, anemia or inflammatorycondition associated with an elevated level of hepcidin. The subjectmay, for example, be a mammal such as a human suffering from Africaniron overload, alpha thalassemia, Alzheimer's disease, anemia, anemia ofcancer, anemia of chronic disease, anemia of inflammation,arteriosclerosis or atherosclerosis (including coronary artery disease,cerebrovascular disease or peripheral occlusive arterial disease),ataxias, ataxias related to iron, atransferrinemia, cancer,ceruloplasmin deficiency, chemotherapy-induced anemia, chronicrenal/kidney disease (in particular anemia associated with chronickidney disease), including end stage renal disease or chronicrenal/kidney failure, cirrhosis of liver, classic hemochromatosis,collagen-induced arthritis (CIA), a condition involving hepcidin excess(elevated hepcidin), congenital dyserythropoietic anemia, congestiveheart failure, Crohn's disease, diabetes, a disorder of ironbiodistribution, a disorder of iron homeostasis, a disorder of ironmetabolism, ferroportin disease, ferroportin mutation hemochromatosis,folate deficiency. Friedrich's ataxia, funicular myelosis, gracilesyndrome, a bacterial infection such as H. pyelori infection,Hallervordan Spatz disease, hemochromatosis, hemochromatosis resultingfrom mutations in transferrin receptor 2, hemoglobinopathies, hepatitis,hepatitis (Brock), hepatitis C, hepatocellular carcinoma, hereditaryhemochromatosis a viral infection such as HIV, Huntington's disease,hyperfernitinemia, hypochromic microcytic anemia, hypoferremia, insulinresistance, iron deficiency anemia, an iron deficiency disorder, an ironoverload disorder, an iron-deficiency condition with hepcidin excess,juvenile hemochromatosis (HFE2), multiple sclerosis, a mutation of agene involved in iron metabolism, for instance expressing a proteininvolved therein such as transferrin receptor 2, HFE, hemojuvelin orferroportin, neonatal hemochromatosis, a neurodegenerative diseaserelated to iron, osteopenia, osteoporosis pancreatitis, Pantothenatekinase-associated neurodegeneration, Parkinson's disease, pellagra,pica, porphyria, porphyria cutanea tarda, pseudoencephalitis, pulmonaryhemosiderosis, a red blood cell disorder, rheumatoid arthritis, sepsis,sideroblastic anemia, systemic lupus erythematosus, thalassemia,thalassemia intermedia, transfusional iron overload, a tumor,vasculitis, vitamin B6 deficiency, vitamin B12 deficiency Wilson'sdisease, or inflammatory condition associated with an elevated level ofhepcidin.

As a further illustrative example a mutein according to the presentinvention can in some embodiments be used in combination witherythropoietin. Anemia in patients with cancer (AC) and anemia ofchronic disease (ACD) are associated with high concentrations ofhepcidin (about 30 nmol/L) leading to serum iron deficiency and thus toreduced erythropoiesis. Subjects with baseline hepcidin concentrationsbelow 13 nmol/L in serum have been reported to show a better response toerythropoietin (EPO) therapy than subjects with concentrations above 13nmol/L. Therefore the treatment of anemic cancer patients with ahepcidin antagonist can improve their response to erythropoietin.

Furthermore a widespread phenomenon among anemic subjects is resistanceto recombinant erythropoietin (rhEPO), a therapeutic problem that can beovercome by combinatorial therapy with a mutein according to the presentinvention. Hepcidin likely plays a major role in this rhEPO resistance.Sasu et al. (Blood (2010) 115, 17, 3816-3624) have shown a distinctcorrelation between increased hepcidin level and resistance toerythropoiesis-stimulating agents (ESAs) in mice. They also were able torestore ESA-responsiveness by the administration of a hepcidin-specificantibody.

In yet another aspect the invention relates to the use of a muteinaccording to the invention in diagnosis. The use of a mutein accordingto the invention is typically for the diagnosis of a disease or disorderassociated with an altered level of a Hepcidin as well as a respectivemethod of diagnosis. The use may in some embodiments involve assessingthe level of a hepcidin in a body fluid of a subject. For this purposebody fluid may have been taken from the respective subject. The level ofthe Hepcidin may be compared to a control sample, which is known toinclude a normal level of the Hepcidin. It may thereby be determinedwhether non-physiological levels of the Hepcidin are present in thesubject.

Accordingly, the invention also relates to a mutein as defined above forthe diagnosis of a disease or disorder associated with an altered, e.g.increased or reduced, level of a Hepcidin. In some embodiments thedisease is an anemia, including, but not limited to, anemia resultingfrom infection, inflammation, chronic disease, and/or cancer. Thedisease or disorder may for instance be associated with decreased levelsof a Hepcidin, such as hereditary hemochromatosis, an iron-loadinganemia or Hepatitis C. The disease or disorder may also be associatedwith increased levels of a Hepcidin, e.g. anemia of inflammation,iron-refractory iron deficiency anemia or a chronic kidney disease.Hepatitis C for instance typically involves a hepatic iron overload,generally via hepcidin synthesis suppression. In the context ofdiagnosis a mutein according to the invention can be used to assesshepcidin levels in body fluid of a subject. Since anemic cancer patientswith low hepcidin concentrations (<13 nmol/L) have been observed to showa better response to erythropoietin therapy than patients with highhepcidin concentrations (>13 nmol/L) hepcidin serum concentrations canfor instance be used for predicting the response to epoetin therapy(about 50% of the patients are EPO resistant).

In still another aspect, the present invention features a diagnostic oranalytical kit comprising a mutein according to the present invention.

The subject in need of such a treatment may be a mammal, such as ahuman, a dog, a mouse, a rat, a pig, an ape such as cynomolgous monkeysto name only a few illustrative examples.

In still another aspect, the present invention features a method for invivo imaging in a subject, including administering to said subject amutein of the invention or a pharmaceutical composition comprising amutein of the invention. The subject may be defined as above.

The invention is further illustrated by the following non-limitingExamples and the attached drawings.

Unless otherwise indicated, established methods of recombinant genetechnology were used, for example, as described in Sambrook at al.(supra).

Example 1 Construction of a Mutant Lcn2 Phage Display Library

A combinatorial library of Lcn2 variants was generated on the basis ofthe cloned cDNA (Breustedt et al. (2006) Biochim. Biophys. Acta 1764,161-173), which carried the amino acid substitutions Cys87Ser, to removethe single unpaired thiol side chain (Goetz et al. (2000) Biochemistry39, 1935-1941), as well as Gln28His to introduce a second BstXIrestriction site. Mutagenesis and polymerase chain reaction (PCR)assembly of this region was essentially performed according to apublished strategy (Beste at al. (1999) Proc. Natl. Acad. Sci. USA 96,1898-1903; Skerra (2001) J. Biotechnol. 74, 257-275), this time using aone pot amplification reaction with oligodeoxynucleotides (sequences ofSEQ ID NO: 16 to SEQ ID NO: 25) as illustrated in FIG. 1.Oligodeoxynucleotides were designed such that the primers with sequencesof SEQ ID NO: 16 to SEQ ID NO: 19 corresponded to the coding strand andcarried degenerate codons at the amino acid positions 36, 40, 41, 49,52, or 68, 70, 72, 73, 77, 79, 81, or 96, 100, 103, 106, or 125, 127,132, 134 respectively, while primers with sequences of SEQ ID NO: 20 toSEQ ID NO: 23 corresponded to the non-coding strand and did not carrydegenerate codons or anticodons. The two flanking primers with SEQ IDNO: 24 and SEQ ID NO: 25 were used in excess and served for theamplification of the assembled randomized gene fragment. All PCR stepswere performed using Go-Taq Hot Start DNA polymerase (Promega, Mannheim,Germany) as described (Schlehuber et al. (2000) J. Mol. Biol. 297,1105-1120).

Oligodeoxynucleotides that did not carry degenerate codons werepurchased in HPLC grade from Metabion (Munich, Germany). NNK-containingoligodeoxynucleotides were purchased desalted from the same vendor andfurther purified by urea PAGE. The resulting DNA library was cut withBstXI (Promega. Mannheim, Germany) and cloned on the phagemid vectorphNGAL102 (SEQ ID NO: 26), which is based on the generic expressionvector pASK111 (Vogt and Skerra (2001) J. Mol. Recognit. 14 (1), 79-86)and codes for a fusion protein composed of the OmpA signal peptide, themodified mature Lcn2, followed by an amber codon, and the C-terminalfragment of the gene III coat protein of the filamentous bacteriophageM13, i.e. similar as previously described for the bilin-binding protein(Beste et al., supra; Skerra, supra). After electroporation of E. coliXL1-Blue (Bullock et al. (1987) Biotechniques 5, 376-378) with theligation mixture of 8.4 μg digested PCR product and 94 μg digestedplasmid DNA, 1×10¹⁰ transformants were obtained.

Alternatively, a cloned synthetic Lcn2 random library, which isdescribed in FIG. 2, was obtained from Sloning BioTechnology GmbH(Puchheim, Germany). The central gene cassette flanked by the two BstXIrestriction sites was amplified via PCR in 20 cycles using appropriateprimers (SEQ ID NO: 24 and SEQ ID NO: 25) and subcloned on phNGAL108(SEQ ID NO: 27), which is based on the generic expression vector pASK75(Skerra (1994) Gene 151, 131-135) and carries essentially the samefeatures as phNGAL102 (SEQ ID NO: 26) but mediates ampicillin resistanceinstead of chloramphenicol resistance, in the same way, yielding alibrary with a complexity corresponding to 1.7×10¹⁰ independenttransformants.

The following steps in library generation were performed identically forboth Lcn2 libraries. 100 ml of the culture, containing the cells whichwere transformed with the phasmid vectors on the basis of phNGAL102 orphNGAL108, respectively, coding for the library of the lipocalin muteinsas phage pIII fusion proteins, were transferred to a sterile Erlenmeyerflask and incubated for one hour at 37° C., 160 rpm in 2YT mediumwithout antibiotic selection pressure. Before infection with VCS-M13helper phage the culture was diluted in 2YT medium to an OD550 of 0.1with the corresponding antibiotic added and further grown underidentical conditions until an OD550 of 0.6 was reached. After infectionwith VCS-M13 helper phage (Agilent Technologies, La Jolla, USA) at amultiplicity of infection of approximately 10 the culture was shaken foradditional 30 min at 37° C., 100 rpm. Then the incubator temperature waslowered to 26° C. and the shaker speed was increased again to 160 rpm,after 10 min kanamycin (70 μg/ml) was added, followed by induction ofgene expression via addition of anhydrotetracycline (ACROS Organics,Geel, Belgium) at 25 μg/l (125 μl of a 200 μg/ml stock solution indimethylformamide. DMF per liter of culture). Incubation continued foranother 12-15 h at 26 C, 160 rpm.

Cells from the complete culture were sedimented by centrifugation (30min. 18000 g, 4° C.). The supernatant containing the phagemid particleswas sterile-filtered (0.45 μm), mixed with ¼ volume 20% w/v PEG 8000,15% w/v NaCl, and incubated on ice for at least 2 h. Aftercentrifugation (30 min. 18000 g, 4° C.) the precipitated phagemidparticles from 1 liter of culture were dissolved in 30 ml of cold BBS/E(200 mM Na-borate, 160 mM NaCl, 1 mM EDTA pH 8.0) containing 50 mMbenzamidine (Sigma) and Pefabloc 1 μg/ml (Roth, Karlsruhe, Germany). Thesolution was incubated on ice for 1 h. After centrifugation ofundissolved components (10 min. 43000 g, 4° C.) each supernatant wastransferred to a new reaction vessel.

Addition of ¼ volume 20% w/v PEG 8000, 15% w/v NaCl and incubation for60 min on ice served to reprecipitate the phagemid particles until thephagemids were aliquoted and frozen at −80° C. for storage. For thefirst selection cycle phagemids were thawed and centrifuged (30 min.34000 g, 4° C.), the supernatant was removed, and the precipitatedphagemid particles were dissolved and combined in a total of 400 μl PBScontaining 50 mM benzamidine. After incubation for 30 min on ice thesolution was centrifuged (5 min, 18500 g, 4° C.) in order to removeresidual aggregates and the supernatant was used directly for the phagedisplay selection.

Example 2 Procurement of Soluble Hepcidin 25 Peptides

Synthetic non-modified Hepcidin-25 (human DTHFPICIFCCGCCHRSKCGMCCKT, SEQID NO: 28, 2789.4 g/mol; mouse DTNFPICIFCCKCCNNSQCGICCKT, SEQ ID NO: 29,2754.2 g/mol; rat DTNFPICLFCCKCCKNSSCGLCCIT, SEQ ID NO: 30, 2711.9g/mol) and the C-terminal biotinylated rat Hepcidin-25(DTNFPICLFCCKCCKNSSCGLCCIT (SEQ ID NO: 30)-Mini-PEG-linker-K-Biotin,3210.5 g/mol) was obtained from PeptaNova GmbH (Sandhausen, GE).

The human and mouse C-terminal biotinylated Hepcidin-25 was obtainedfrom Bachem AG (Bubendorf, CH). Analogous to the rat Hepcidin-25 thesetargets were biotinylated via a Lysine residue coupled to the C-terminusvia a Mini-PEG linker.

Example 3 Generation of a Library with 10 Billion Independent NGALMuteins

A random library of NGAL lipocalin (Lcn2) with high complexity wasprepared essentially as described in Example 1 above. The amplificationreaction is illustrated in FIG. 1, the phagemid vector phNGAL102 is ofSEQ ID NO: 26.

Example 4 Phagemid Presentation and Selection of NGAL Muteins withAffinity for Human Hepcidin

Phagemid display and selection was performed employing the phagemidsobtained from Example 1 essentially as described in international patentapplication WO/20051019256. The library was subjected to 3 cycles ofphage display selection against the soluble. C-terminal biotinylatedhuman Hepcidin-25 target peptide.

2×10¹² to 1×10¹³ phagemids of the library obtained in Example 1 wereused. In brief, the phagemids were centrifuged (21460×g, 4° C., 20 min)and resuspended 1 ml PBS (4 mM KHaPO₄, 16 mM Na₂BPO₄, 115 mM NaCl, pH7.4) containing 50 mM benzamidine. PBS containing 1% w/v Casein (Sigma)and 0.1% Tween 20® was used as blocking buffer. Prior to the incubationwith the target protein, phagemids from the library were incubated withcasein-blocked Streptavidin beads for 30 minutes for the depletion ofphagemids representing multi-reactive or misfolded lipocalin mutein orStreptavidin bead-specific muteins.

In different Panning approaches a 1 μM solution of target was eithercaptured on Streptavidin™-coated, 1% Casein-blocked magnetic beads priorto the incubation with phagemids (solid in solution approach) or 500 nMHepcidin-25 was incubated in solution with 3·10¹² phagemids from theNGAL library blocked with 1% Casein (solution approach). In the solutionapproach peptide bound phagemids were captured via Streptavidin™-coatedmagnetic beads (Invitrogen/Dynal) within 20 min, followed by 8 washcycles and elution with either 300 μL 70 mM Triethylamin for 10 min, andneutralization with an appropriate amount of 1 M Tris/HCl, pH 7.4 (basicelution) or with 300 μL 0.1 M Glycin/HCl pH 2.2 for 10 min, andneutralization with an appropriate amount of 0.5 M Tris-Base (acidicelution).

In the solid in solution approach blocked phagemids were incubated withthe Streptavidin bead-coated target followed by 8 wash cycles andelution as described above [0199]. Beginning with the second enrichmentcycle, only half of the combined phagemid solutions were used forphagemid amplification.

Phagemid amplification between each panning cycle was performed asdescribed in Schlehuber, S. et al. (J. Mol. Biol. (2000), 297,1105-1120).

Two further selection rounds against Hepcidin-25 were carried out inthis way employing the preparation of amplified phagemids from therespective previous enrichment cycle with the exception that only about1×10^(I1) phagemids were utilized beginning with the second enrichmentcycle.

Example 5 Identification of hHepcidin-Specific Muteins UsingHigh-Throughput ELISA Screening

Screening of the muteins selected according to Example 4 was performedessentially as described in Example 3 of international patentapplication WO 2006/56464.

Lipocalin muteins were selected in a HT-screening ELISA. Therein, NGALvariants equipped with a T7 detection tag (Novagen) as well as aStrep-tag II affinity tag (IBA) were soluble expressed in a 96 wellmicrotiter plate using the E. coli strain TG1/F with phNGAL 101. Thisvector corresponds to phNGAL 98 (SEQ ID NO: 31) with an N-terminal T7tag consisting of 11 amino acids (MASMTGGQQMG) (SEQ ID NO: 34, see alsoFIG. 4B). Lipocalin mutein expression was induced overnight at 22° C. at700 rpm with anhydrotetracycline (0.2 μg/ml) at an OD₅₅₀ of 0.6.Afterwards, cells were lysed (100 mM Na-borate, pH 8.0, 80 mM NaCl, 1 mMEDTA, 0.025% w/v lysozyme) for 1 h under agitation. To minimizenon-specific binding in the subsequent ELISA screen, the crude celllysates were supplemented with 2% w/v BSA and 0.1% v/v Tween 20 andtested in ELISA for binding to human Hepcidin-25. Therefore, solubleC-terminal biotinylated human Hepcidin-25 was immobilized on wells ofblack Fluotrac 600 ELISA plates (Greiner; 384 well) with 1 μg/ml viacapturing by Neutravidin (5 μg/ml, Thermo Scientific). Neutravidin,Streptavidin, 5 μg/ml each, and 3% milk were used as negative control.Plates were blocked with PBST/0.1 containing 2% w/v BSA, andsubsequently incubated with the bacterial cell extract for 1 h at roomtemperature plates were washed five times and bound Lipocalin muteinswere detected via an anti-T7 monoclonal antibody-HRP conjugate(Novagen), diluted 1:10.000 in PBST/O 1. Therefore, QuantaBlu™ (Pierce;1:2 diluted in PBS/T 0.1%) was used as fluorogenic HRP substrate. After45 min of signal development at room temperature fluorescence wasexcited at a wavelength of 320 nm (±12.5 nm) and measured at 430 nm(±17.5 nm) in a GENiosPlus plate reader (Tecan).

In a reverse ELISA approach soluble expressed muteins from the crudecell lysate were captured in ELISA plates via their T7-tag followingincubation with varying amounts of C-terminal biotinylated hHepcidin toreach target-limiting conditions in order to differentiate the muteinsby their affinity. Binding of the target was detected via Extravidin-HRPconjugate (Sigma). One could compete for mutein binding by the additionof 100 nM non-biotinylated human Hepcidin-25 indicating, that themuteins bind the non-modified hHepcidin-25 as well.

Screening of 2160 clones, selected as described in Example 4, led to theidentification of more then 1000 primary hits indicating the successfulisolation of target-specific muteins. The reverse ELISA approach undertarget-limiting conditions and the competition ELISA allowed for adifferentiation of hepcidin-specific muteins in terms of their targetaffinity. Using these ELISA approaches the clones with SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQID NO: 12 were identified. The sequences of these muteins are depictedin FIG. 3.

Example 6 Production of Hepcidin-Binding Muteins (NGAL)

The recombinant Lcn2 and the hHepcidin-specific Lcn2 variants wereproduced by periplasmic secretion in E. coli K12 strain JM83(Yanisch-Perron et al. (1985) Gene 33, 103-119), the E. coli supE strainTG1˜F˜ (a derivative of E. coli K12 TG1 [Kim et al. (2009) J. Am. Chem.Soc. 131, 3565-3576] that was cured from its episome using acridiniumorange), E. coli BL21 (Studier and Moffat (1986) J. Mol. Biol. 189,113-130), or E. coli W3110 (Bachmann (1990) Microbiol. Rev. 54,130-197).

For a small scale soluble protein expression the plasmid phNGAL98 (SEQID NO: 31) was used, encoding a fusion of the OmpA signal peptide withthe respective mutein and the C-terminal Strep-tag II, whereby theplasmid carries the two non-compatible BstXI restriction sites forunidirectional subcloning of the mutated gene cassette. Growth wasallowed to occur in a 2 L shaking flask culture in the presence ofLB-Ampicillin medium according to the protocol described in Schlehuber,S. et al. (J. Mol. Biol. (2000), 297, 1105-1120). For larger amounts ofprotein the periplasmatic production was performed with the same vectorexpressed in the E. coli strain W3110 via bench top fermentercultivation in a 1 l or 10 l vessel based on the protocol described inSchiweck, W., and Skerra, A. Proteins (1995) 23, 581-565).

In order to increase the in vivo half-life, selected lipocalin muteinswere exemplarily modified by the following procedures.

An ABD-fusion protein was constructed and periplasmatically expressedfor the mutein of SEQ ID NO: 1 The albumin binding domain from thestreptococcal protein G was fused to the C-terminus of the mutein viathe original linker derived from streptococcal protein G as described inSEQ ID NO: 15.

In the case of site-directed PEGylation the hNGAL muteins having a freecystein residue at amino acid position 87 (SEQ ID NO 13, SEQ ID NO: 14)were used for PEGylation with branched 40 k PEG-maleimide. To this aim,the Serine at position 87 was back-mutated to a Cysteine that originallyoccurs in hNGAL wildtype by site-directed mutagenesis (Quick-changemutagenesis Kit, Stratagene). Prior to the PEGylation reaction the freecysteine residue was reduced in a 1:1 molar ratio of Anticalin with TCEPfor 3 h at RT. Thereafter, PEGylation was performed by mixing theprotein with >2 molar excess of PEG40-maleimide reagent for 1.5 h at RT.

The Lcn2 variants were purified from the periplasmic fraction in asingle step via streptavidin affinity chromatography (Strep-Tactin™Superflow, IBA) using a column of appropriate bed volume according tothe procedure described by Skerra, A. & Schmidt, T. G. M. (2000) (Use ofthe Strep-tag and streptavidin for detection and purification ofrecombinant proteins. Methods Enzymol. 326A, 271-304). To achieve higherpurity and to remove any aggregated recombinant protein, a gelfiltration of the muteins was finally carried out on a Superdex 75 HR10/30 column (24-ml bed volume, Amersham Pharmacia Biotech, Freiburg,Germany) in the presence of PBS buffer. The monomeric protein fractionswere pooled, analysed for purity by SDS-PAGE (Fling and Gregerson (1986)Anal. Biochem. 155, 83-88), and used for further biochemicalcharacterization.

The pegylated versions of hNGAL muteins were purified by chromatographyand, where necessary, a further reduction of bacterial endotoxins wasachieved by MustangE membrane (Pail Corporation, US) filtration.

Example 7 Affinity Measurement Using ELISA Techniques

A “direct” ELISA was performed to verify the binding affinity andspecificity of the selected Lcn2 muteins. Therefore, a constantconcentration of 1 μg/ml C-terminal biotinylated hepcidin (Bachem AG,CH) was captured on the surface of a polystyrol plate (Greiner, GE) viaNeutravidin (Thermo Scientific, 5 μg/ml). Two step dilution series ofpurified Lcn2 muteins were incubated with the captured hepcidin for h atroom temperature and detected either via the Strep-tag II using a rabbitanti-strep-tag II polyconal antibody (GenScript, USA) or by using ascaffold-specific polyclonal rabbit antibody. In both cases an antirabbit IgG-HRP conjugate (Abcam, UK) was employed as secondary detectionantibody.

The absorption ΔA at 320 nm was measured in an ELISA reader (Tecan, GE)and the data were fitted with Graphpad Prism software (Statcom, USA).

Results from measurements employing the muteins of the sequences of SEQID NO: 1 to SEQ ID NO: 12, as well as of SEQ ID NO: 14, linked to PEG40,of SEQ ID NO: 13, linked to PEG40 and of SEQ ID NO: 1, linked toalbumine-binding domain (ABD) (SEQ ID NO 15) are summarized in FIG. 5.

K_(D) values of the selected Lcn2 muteins vary from 220 pM up to 8.8 nM.All muteins bound human and cynomolgus hepcidin-25 with comparableaffinity. Serum half-life extension of the lipocalin mutein of SEQ IDNO: 1 via C-terminal fusion of the albumine-binding domain had nosignificant effect on the binding affinity of the mutein whereaspegylation reduced the binding affinities in this ELISA formatsignificantly by a factor of 5 for SEQ ID NO: 8 and a factor of 8 forthe mutein of SEQ ID NO: 1.

The binding affinity of the Lcn2 muteins to non-modified hepcidin-25 insolution was evaluated in a competition ELISA approach. Therefore, aconstant concentration of 1 μg/ml C-terminal biotinylated human hepcidin(Bachem AG, CH) was captured on the surface of a polystyrol plate(Greiner, GE) via Neutravidin (Thermo Scientific, 5 μg/ml, GE). Inparallel a two step dilution series of non-biotinylated human hepcidinstarting from 1 μM was incubated with a constant concentration ofhepcidin-specific mutein for 1 h at room temperature in a non-proteinbinding 96 well polypropylene plate (Nunc, GE). The constantconcentration of lipocalin muteins corresponds to the EC₅₀ of therespective muteins as determined in a direct ELISA as described above inthis example. In the following the mixture of non-modified humanhepcidin and lipocalin mutein was transferred onto the hepcidin-capturedNeutravidin plate. The C-terminal biotinylated hepcidin was allowed tocompete with the non-modified hepcidin for Anticalin binding for 20 minat room temperature. During these 20 min. free lipcocalin mutein wasbound to the captured hepcidin and detected via a rabbit anti-strep-tagII polyconal antibody (GenScript, USA). A goat anti-rabbit IgG-HRPconjugate (Abcam, UK) was employed as secondary detection antibody.Parallel to the competition assay, anticalin binding was determined onthe same plate in a “direct” ELISA, in order to obtain a standard curvelinking the RFU values to anticalin concentration. This curve was thenused to standardize competition data to the level of anticalins boundsto the plate and fitted with Graphpad software IC₅₀ values correspond tothe half-maximum amount of lipocalin mutein bound to the plate.

Results from measurements employing the muteins of the sequences of SEQID NO: 1 to SEQ ID NO: 12, as well as of SEQ ID NO: 14, linked to PEG40,of SEQ ID NO: 13, linked to PEG40 and of SEQ ID NO: 1, linked to ABD(SEQ ID NO: 15), are summarized in FIG. 6.

IC₅₀ values of the selected Lcn2 muteins vary from 100 pM up to 10.8 nM.Serum half-life extension via the albumine-binding domain had no effecton the binding affinity of the mutein of SEQ ID NO: 1, whereaspegylation reduced the binding affinities by a factor of 2 for SEQ IDNO: 13-PEG40 and by a factor of 4 for SEQ ID NO: 14-PEG40, respectively.

Example 8 Affinity Measurement Using Surface-Plasmon-Resonance (SPR)

Surface plasmon resonance was used to measure binding kinetics andaffinity of the lipocalin muteins disclosed herein.

Lipocalin muteins were immobilized to a CM5 sensor chip using standardamine chemistry. The surface of the chip was activated using EDC andNHS. Subsequently, 20 μg/mL lipocalin mutein solutions in 10 mM sodiumacetate pH 4.5 (60 μg/mL in 10 mM sodium acetate pH 4.0 for peglytedlipocalin mutein) were applied at a flow rate of 5 μL/min until asurface density of 500-700 resonance units (RU) for non-modifiedlipocalin muteins and of approximately 1600 RU for pegylated lipocalinwith the sequence of SEQ ID NO: 13 was achieved. Residual activatedgroups were saturated with ethanolamine. The reference channels weretreated with EDC/NHS following ethanolamine (blank immobilization). Allreagents and materials were purchased from GE Healthcare.

Serial dilutions of human and cynomolgus Hepcidin-25 in running buffer(HBS-EP+, GE Healthcare, BR-1006-68) were applied to the preparedsurface. The following parameters were used for the binding assay,contact time 60 s, dissociation time 180-360 s, flow rate 30 μL/min. Allmeasurements were performed on a Biacore T100 instrument (GE Healthcare)at 25° C. Regeneration of the surfaces having lipocalins immobilizedthereon were achieved with subsequent injections of 2 M/4 MGuanidinium-HCl (120-600 s) and 10 mM glycine-HCl pH 1.5/2.0 (40-240 s)followed by an extra wash with running buffer and a stabilization periodof 120 s.

Data were evaluated with Biacore T100 Evaluation software (V 2.0.1).Double referencing was used. The 1:1 Binding model (Langmuir) was usedto fit the raw data.

Dublicates were reproducible and no binding to the reference channel wasdetected. The binding parameters of the lipocalin muteins with thesequences of SEQ ID NO: 1. SEQ ID NO: 8 as well as of SEQ ID NO: 13,linked to PEG40, to human and cynomolgus Hepcidin-25 are summarized inFIG. 7.

Cynomolgus Hepcidin-25 was binding to immobilized lipocalin muteins withan approximately 2-fold higher affinity compared to the human target.Kinetic analysis of hHepcidin-25 on the immobilized pegylated varianthaving the sequence of SEQ ID NO. 13 revealed a high affinity of 40 pM.

Example 9 Cell-Based Assay for Hepcidin-Induced Internalization andDegradation of Ferroportin

An in vitro cell-based assay was used to measure the neutralizationactivity of the lipocalin muteins of the present invention that aredirected against human hepcidin. The assay is based on hepcidin-inducedinternalisation and degradation of its receptor, ferroportin and wasimplemented basically as described (Nemeth et al, 2004, 2006).

Briefly, a HEK-293 stable cell line was prepared that allowed for theinducible expression of murine ferroportin (FPN) carboxy-terminallyfused with green fluorescent protein (GFP). The inducible expression ofthe FPN-GFP fusion protein was controlled by Doxycyclin using thecommercially available tetracycline-regulated T-REx expression system(Invitrogen, Karlsruhe, Germany). The FPN-GFP coding sequence was clonedinto pcDNA 4/TO vector, which contains an inducible promoter and aZeocin resistance marker. The resulting construct was stably transfectedinto T-REx-293 cells which express the regulatory protein required fordoxycycline-inducible expression.

The assay for hepcidin-induced internalisation of the hepcidin receptorwas performed as follows: Cells of the T-REx-293::FPN-GFP stable linewere seeded in T75 cell culture flasks at 80% confluence. In the eveningFPN-GFP expression was induced with 4 ng/ml Doxycyclin and stabilizedwith 10 μM Ammonium iron (III) citrate for 16 h at 37° C. On the nextmorning cells were trypsinized and seeded in a 24-well plate at 0.3million cells/well in a volume of 450 μl. Cells were allowed to attachfor 1 h at 37° C. prior to the addition of hepcidin. Cells wereincubated at 37° C. for 24 h and GFP fluorescence of the detached cellsuspension was analyzed by flow cytometry.

The EC80 (40 nM) of hepcidin-mediated degradation of the Fpn-GFP fusionprotein was used in neutralization assays. For this purpose Anticalinswere incubated with hepcidin at room temperature for 30 min prior toaddition to the cells. Following the 24 h incubation period fluorescencewas quantified as described above.

The anti-hepcidin lipocalin muteins having the sequences of SEQ ID NO.1, SEQ ID NO. 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO. 10, SEQ ID NO: 11, as well as of SEQ ID NO: 14 linked toPEG40 and SEQ ID NO: 13 linked to PEG40 and SEQ ID NO: 1 linked to ABD(SEQ ID NO: 15) neutralized the bioactivity of human hepcidin-25 withIC50 values shown in FIG. 8.

Example 10 Anti-Hepcidin Lipocalin Muteins Neutralize Human Hepcidin inMice

The activity of anti-human hepcidin lipocalin muteins was evaluated invivo in mice that were administered human hepcidin in an amountsufficient to generate a hypoferremic response as described (Nemeth etal (2006) Blood, 107:328-333).

Two weeks before the experiment C57BL/6 mice were switched to an irondeficient diet to suppress endogenous hepcidin. Prior to the experimenta 3-fold molar excess of lipocalin mutein was allowed to bind synthetichuman hepcidin-25 for 30 minutes. In parallel wildtype lipocalin (NGAL98) was pre-incubated with human hepcidin-25 in the same molar ratio asan isotypic control. Mice received a single intraperitoneal (i.p.)injection of either PBS (vehicle) or 2 mg/kg hepcidin or 2 mghepcidin/Kg pre-incubated with either lipocalin mutein or wild typelipocalin (negative control). Two hours later, blood was collected underisoflurane anesthesia and total serum iron levels were determined usinga colorimetric assay on a KoneLab XTi clinical analyzer.

The results are depicted in FIG. 9 as total serum iron levels in μMconcentrations. Hepcidin treatment induced a significant drop of serumiron levels in iron-starved mice. Hepcidin pre-incubated with thewildtype lipocalin also exhibited hypoferremia. The pre-complexation ofhuman hepcidin with the lipocalin mutein protected the animals from thehypoferremic response.

Example 11 Determination of Pharmacokinetic (PK) Parameter forAnti-Hepcidin-25 Lipocalin Muteins

Pharmacokinetic (PK) parameters (half-life plasma concentration) for theLcn 2 mutein having the sequence of SEQ ID NO: 14 linked to PEG40 and ofSEQ ID NO: 1 linked to ABD (SEQ ID NO: 15) were determined followingi.v. single bolus administration in NMRI mice and Cynomolgus (Macaccafascicularis) at doses depicted in FIG. 10. Plasma was prepared fromterminal blood samples taken at pre-determined timepoints and theconcentrations of the lipocalin mutein were determined by ELISA. Theelimination rate constant was calculated by least squares linearregression of the terminal portion of the log transformed plasmaconcentration-time curve. The start of the terminal elimination phasefor each individual profile was defined by visual inspection and was thefirst point at which there was no systematic deviation from thelog-linear decline in serum concentrations. T½ was calculated accordingto the following formula:

$t_{1/2} = \frac{\ln (2)}{\lambda_{x}}$

T½ SEQ ID NO: 14-PEG (mouse): 27.9 h; T½ SEQ ID NO: 1-ABD (mouse): 30 h:T½ SEQ ID NO: 14-PEG (Cyno): 88 h.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Further, itwill be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Thecompositions, methods, procedures, treatments, molecules and specificcompounds described herein are presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention are defined by the scope of the claims. The listing ordiscussion of a previously published document in this specificationshould not necessarily be taken as an acknowledgement that the documentis part of the state of the art or is common general knowledge.

It must be noted that as used herein, the singular forms “a”. “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “an antibody” includes one ormore of such different antibodies and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or” as used herein.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising. “including,” containing,” etc. shall be read expansivelyand without limitation. In each instance herein any of the terms“comprising”, “consisting essentially of” and “consisting of” may bereplaced with either of the other two terms.

Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of theinventions embodied therein herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

<160> NUMBER OF SEQ ID NOS: 48 <210> SEQ ID NO 1 <211> LENGTH: 178<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 1Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 2 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 2Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Thr Ala Gly Asn Ser Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Gln Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Arg Val Phe Phe Glu Gly Lys Lys Cys Arg Tyr Val Ile 65                  70                  75                  80  Glu Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys                 85                  90                  95      Ile Lys Ser Ala Pro Gly Gly Thr Ser Ile Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Leu Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 3 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 3Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Val Ala Gly Asn Gly Leu Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Leu Lys Met His Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Arg Val Leu Phe Val Arg Lys Lys Cys Arg Tyr Tyr Ile 65                  70                  75                  80  Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg                 85                  90                  95      Ile Lys Ser Glu Pro Gly Arg Thr Ser Phe Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Met Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 4 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 4Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Val Ala Gly Asn Glu Met Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Leu Lys Met Leu Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Arg Val Met Phe Glu Tyr Lys Lys Cys Val Tyr Leu Ile 65                  70                  75                  80  Glu Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Thr                 85                  90                  95      Ile Lys Ser Val Pro Gly Leu Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Arg Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 5 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 5Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Ala Ala Gly Asn Ser Leu Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Arg Val Asn Phe Gly Gly Lys Lys Cys Ser Tyr Leu Ile 65                  70                  75                  80  Glu Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Ser                 85                  90                  95      Ile Lys Ser Arg Pro Gly Ala Thr Ser Val Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Leu Val Thr Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 6 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 6Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Leu Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Arg Val Gln Phe Gly Glu Lys Lys Cys Gly Tyr Gly Ile 65                  70                  75                  80  Glu Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Ser                 85                  90                  95      Ile Lys Ser Val Pro Gly Gly Thr Ser Arg Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 7 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 7Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Arg Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Gln Lys Met Phe Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Gly Val Asp Phe Arg Thr Lys Lys Cys Leu Tyr Ser Ile 65                  70                  75                  80  Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Val                 85                  90                  95      Ile Lys Ser Gln Pro Gly Trp Thr Ser Tyr Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 8 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 8Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Pro Leu Ala Glu Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Gly                 85                  90                  95      Ile Lys Ser Gly Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Val Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 9 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 9Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Ser Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Gly Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 10 <211> LENGTH: 179 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 10Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Arg Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Pro Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly Phe  <210> SEQ ID NO 11 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 11Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Gly Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 12 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 12Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Glu Glu Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Lys Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 13 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 13Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Pro Leu Ala Glu Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Gly                 85                  90                  95      Ile Lys Ser Gly Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Val Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 14 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL <400> SEQUENCE: 14Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 15 <211> LENGTH: 243 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Mutein of hNGAL with ABD domain and STREP tag <400> SEQUENCE: 15Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65                  70                  75                  80  Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln         115                 120                 125             Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly Ser Ala Gly Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys             180                 185                 190         Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr         195                 200                 205             Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu     210                 215                 220                 Ile Asp Glu Ile Leu Ala Ala Leu Pro Ser Ala Trp Ser His Pro Gln 225                 230                 235                 240 Phe Glu Lys  <210> SEQ ID NO 16 <211> LENGTH: 88 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      NNK oligomer for positions 36, 40, 41, 49, 52 <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (20)..(21)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (32)..(33)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (35)..(36)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (59)..(60)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (68)..(69)<223> OTHER INFORMATION: a, c, g, or t <400> SEQUENCE: 16gaagtggtat gtggtaggtn nkgcagggaa tnnknnkctc agagaagaca aagacccgnn     60kaagatgnnk gccaccatct atgagctg                                        88<210> SEQ ID NO 17 <211> LENGTH: 79 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      NNK oligomer for positions 68, 70, 72, 73, 77, 79, 81<220> FEATURE:  <221> NAME/KEY: modified_base <222> LOCATION: (20)..(21)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (26)..(27)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (32)..(33)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (35)..(36)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (47)..(48)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (53)..(54)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (59)..(60)<223> OTHER INFORMATION: a, c, g, or t <400> SEQUENCE: 17caagagctac aatgtcaccn nkgtcnnktt tnnknnkaag aagtgtnnkt acnnkatcnn     60kacttttgtt ccaggttcc                                                  79<210> SEQ ID NO 18 <211> LENGTH: 68 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      NNK oligomer for positions 96, 100, 103, 106 <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (19)..(20)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (31)..(32)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (40)..(41)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (49)..(50)<223> OTHER INFORMATION: a, c, g, or t <400> SEQUENCE: 18ggcgagttca cgctgggcnn kattaagagt nnkcctggan nkacgagtnn kctcgtccga     60gtggtgag                                                              68<210> SEQ ID NO 19 <211> LENGTH: 68 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      NNK oligomer for positions 125, 127, 132, 134 <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (19)..(20)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (25)..(26)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (40)..(41)<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (46)..(47)<223> OTHER INFORMATION: a, c, g, or t <400> SEQUENCE: 19gctatggtgt tcttcaagnn kgttnnkcaa aacagggagn nkttcnnkat caccctctac     60gggagaac                                                              68<210> SEQ ID NO 20 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      PCR-Primer with fixed nucleotide sequences      corresponding to the non-coding strand <400> SEQUENCE: 20ggtgacattg tagctcttgt cttctttcag ctcatagatg gtggc                     45<210> SEQ ID NO 21 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      PCR-Primer with fixed nucleotide sequences      corresponding to the non-coding strand <400> SEQUENCE: 21gcccagcgtg aactcgcctg gctgggaacc tggaacaaaa gt                        42<210> SEQ ID NO 22 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      PCR-Primer with fixed nucleotide sequences      corresponding to the non-coding strand <400> SEQUENCE: 22cttgaagaac accatagcat gctggttgta gttggtgctc accactcgga cgag           54<210> SEQ ID NO 23 <211> LENGTH: 64 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      PCR-Primer with fixed nucleotide sequences      corresponding to the non-coding strand <400> SEQUENCE: 23ggagaagcgg atgaagttct cctttagttc cgaagtcagc tccttggttc tcccgtagag     60ggtg                                                                  64<210> SEQ ID NO 24 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      5′ flanking PCR-Oligo biotinylated <400> SEQUENCE: 24ccaggacaac caattccatg ggaagtggta tgtggtaggt                           40<210> SEQ ID NO 25 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      3′ flanking PCR-Oligo biotinylated <400> SEQUENCE: 25ttcagggagg cccagagatt tggagaagcg gatgaagttc                           40<210> SEQ ID NO 26 <211> LENGTH: 4746 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Phage display vector phNGAL102 with CamR used      as backbone for NNK-Library <400> SEQUENCE: 26ccataacgct cggttgccgc cgggcgtttt ttattggcca gatgattaat tcctaatttt     60tgttgacact ctatcattgg tagagttatt ttaccactcc ctatcagtga tagagaaaag    120tgaaatgaat agttcgacaa aaatctagat aacgagggca aaaaatgaaa aagacagcta    180tcgcgattgc agtggctctg gctggcttcg ctaccgtagc gcaggcccag gactccacct    240cagacctgat cccagcccca cctctgagca aggtccctct gcagcagaac ttccaggaca    300accaattcca tgggaagtgg tatgtggtag gtctcgcagg gaatgcaatt ctcagagaag    360acaaagaccc gcaaaagatg tatgccacca tctatgagct gaaagaagac aagagctaca    420atgtcacctc cgtcctgttt aggaaaaaga agtgtgacta ctggatcagg acttttgttc    480caggttccca gccaggcgag ttcacgctgg gcaacattaa gagttaccct ggattaacga    540gttacctcgt ccgagtggtg agcaccaact acaaccagca tgctatggtg ttcttcaaga    600aagtttctca aaacagggag tacttcaaga tcaccctcta cgggagaacc aaggagctga    660cttcggaact aaaggagaac ttcatccgct tctccaaatc tctgggcctc cctgaaaacc    720acatcgtctt ccctgtccca atcgaccagt gtatcgacgg cagcgctggt ggggcctaga    780ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag    840acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg    900ttgtagtttg tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg    960ctatccctga aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg   1020agggtggcgg tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata   1080tcaaccctct cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc   1140cttctcttga ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa   1200ataggcaggg ggcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta   1260aaacttatta ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg   1320gtaaattcag agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat   1380atcaaggcca atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg   1440gtggttctgg tggcggctct gagggtggtg gctctgtggg tggcggttct gagggtggcg   1500gctctgaggg aggcggttcc ggtggtggct ctggttccgg tgattttgat tatgaaaaga   1560tggcaaacgc taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg   1620acgctaaagg caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca   1680ttggtgacgt ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta   1740attcccaaat ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc   1800aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta   1860aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt   1920ttcttttata tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta   1980ataaggagtc ttaataagct tgacctgtga agtgaaaaat ggcgcacatt gtgcgacatt   2040ttttttgtct gccgtttacc gctactgcgt cacggatctc cacgcgccct gtagcggcgc   2100attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct   2160agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg   2220tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga   2280ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt   2340ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg   2400aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc   2460ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat   2520ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat   2580aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa   2640aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga   2700acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga   2760tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat   2820tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg   2880tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga   2940aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata   3000ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga   3060gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt   3120ggccaatatg gacaacttct tcgcccccgt tttcactatg ggcaaatatt atacgcaagg   3180cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca   3240tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta   3300ataggaatta atgatgtctc gtttagataa aagtaaagtg attaacagcg cattagagct   3360gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt   3420agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc   3480cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca   3540agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg   3600agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca   3660attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc   3720agtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga   3780agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt   3840atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg   3900attagaaaaa caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg   3960atggtaactt cactagttta aaaggatcta ggtgaagatc ctttttgata atctcatgac   4020caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa   4080aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc   4140accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt   4200aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg   4260ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc   4320agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt   4380accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga   4440gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct   4500tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg   4560cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca   4620cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa   4680cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgac   4740ccgaca                                                              4746<210> SEQ ID NO 27 <211> LENGTH: 4963 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Phage display vector phNGAL108 with AmpR used      for Sloning Library <400> SEQUENCE: 27ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt     60tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct    120agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc tctggctggc    180ttcgctaccg tagcgcaggc ccaggactcc acctcagacc tgatcccagc cccacctctg    240agcaaggtcc ctctgcagca gaacttccag gacaaccaat tccatgggaa gtggtatgtg    300gtaggtctcg cagggaatgc aattctcaga gaagacaaag acccgcaaaa gatgtatgcc    360accatctatg agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa    420aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg    480ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt ggtgagcacc    540aactacaacc agcatgctat ggtgttcttc aagaaagttt ctcaaaacag ggagtacttc    600aagatcaccc tctacgggag aaccaaggag ctgacttcgg aactaaagga gaacttcatc    660cgcttctcca aatctctggg cctccctgaa aaccacatcg tcttccctgt cccaatcgac    720cagtgtatcg acggcagcgc ttggcgtcac ccgcagttcg gtggggccta gactgttgaa    780agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa    840actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt    900tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct    960gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggttc tgagggtggc   1020ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct   1080ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt   1140gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag   1200ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat   1260taccagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc   1320agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc   1380caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct   1440ggtggcggct ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg cggctctgag   1500ggaggcggtt ccggtggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac   1560gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa   1620ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac   1680gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa   1740atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta   1800ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat   1860gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta   1920tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag   1980tcttaataag cttgacctgt gaagtgaaaa atggcgcaca ttgtgcgaca ttttttttgt   2040ctgccgttta ccgctactgc gtcacggatc tccacgcgcc ctgtagcggc gcattaagcg   2100cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg   2160ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc   2220taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa   2280aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc   2340ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac   2400tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt   2460ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc   2520ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt   2580tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat   2640aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt   2700ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg   2760ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga   2820tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc   2880tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac   2940actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg   3000gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca   3060acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg   3120gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg   3180acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg   3240gcgaactact tactctagct tcccggcaac aattgataga ctggatggag gcggataaag   3300ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg   3360gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat ggtaagccct   3420cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac   3480agatcgctga gataggtgcc tcactgatta agcattggta ggaattaatg atgtctcgtt   3540tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag   3600gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc   3660atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc   3720atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta   3780aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac   3840ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag   3900gtttttcact agagaatgca ttatatgcac tcagcgcagt ggggcatttt actttaggtt   3960gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg   4020atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc   4080cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg   4140aaagtgggtc ttaaaagcag cataaccttt ttccgtgatg gtaacttcac tagtttaaaa   4200ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt   4260cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt   4320ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt   4380tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga   4440taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag   4500caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata   4560agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg   4620gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga   4680gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca   4740ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa   4800acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt   4860tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac   4920ggttcctggc cttttgctgg ccttttgctc acatgacccg aca                     4963<210> SEQ ID NO 28 <211> LENGTH: 25 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: Hepcidin-25 <400> SEQUENCE: 28Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg 1               5                   10                  15      Ser Lys Cys Gly Met Cys Cys Lys Thr             20                  25   <210> SEQ ID NO 29 <211> LENGTH: 25<212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE: <223> OTHER INFORMATION: Hepcidin-25 <400> SEQUENCE: 29Asp Thr Asn Phe Pro Ile Cys Ile Phe Cys Cys Lys Cys Cys Asn Asn 1               5                   10                  15      Ser Gln Cys Gly Ile Cys Cys Lys Thr             20                  25   <210> SEQ ID NO 30 <211> LENGTH: 25<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <220> FEATURE: <223> OTHER INFORMATION: Hepcidin-25 <400> SEQUENCE: 30Asp Thr Asn Phe Pro Ile Cys Leu Phe Cys Cys Lys Cys Cys Lys Asn 1               5                   10                  15      Ser Ser Cys Gly Leu Cys Cys Ile Thr             20                  25   <210> SEQ ID NO 31<211> LENGTH: 3745 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      Expression vector phNGAL98 with AmpR encoding      wild type Lcn2 with the C-terminal Strep-tagII <400> SEQUENCE: 31ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt     60tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct    120agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc tctggctggc    180ttcgctaccg tagcgcaggc ccaggactcc acctcagacc tgatcccagc cccacctctg    240agcaaggtcc ctctgcagca gaacttccag gacaaccaat tccatgggaa gtggtatgtg    300gtaggtctcg cagggaatgc aattctcaga gaagacaaag acccgcaaaa gatgtatgcc    360accatctatg agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa    420aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg    480ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt ggtgagcacc    540aactacaacc agcatgctat ggtgttcttc aagaaagttt ctcaaaacag ggagtacttc    600aagatcaccc tctacgggag aaccaaggag ctgacttcgg aactaaagga gaacttcatc    660cgcttctcca aatctctggg cctccctgaa aaccacatcg tcttccctgt cccaatcgac    720cagtgtatcg acggcagcgc ttggtctcac ccgcagttcg aaaaataata agcttgacct    780gtgaagtgaa aaatggcgca cattgtgcga catttttttt gtctgccgtt taccgctact    840gcgtcacgga tctccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta    900cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc    960cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt   1020tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg   1080gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca   1140cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct   1200attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga   1260tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca   1320cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata   1380tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga   1440gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc   1500ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg   1560cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc   1620ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat   1680cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact   1740tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat   1800tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga   1860tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc   1920ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga   1980tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag   2040cttcccggca acaattgata gactggatgg aggcggataa agttgcagga ccacttctgc   2100gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggct   2160ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct   2220acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg   2280cctcactgat taagcattgg taggaattaa tgatgtctcg tttagataaa agtaaagtga   2340ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac   2400tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg   2460ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt   2520tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt   2580tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt   2640atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg   2700cattatatgc actcagcgca gtggggcatt ttactttagg ttgcgtattg gaagatcaag   2760agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat   2820tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc   2880ttgaattgat catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcttaaaagc   2940agcataacct ttttccgtga tggtaacttc actagtttaa aaggatctag gtgaagatcc   3000tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag   3060accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct   3120gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac   3180caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc   3240tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg   3300ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt   3360tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt   3420gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc   3480tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca   3540gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata   3600gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg   3660ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct   3720ggccttttgc tcacatgacc cgaca                                         3745<210> SEQ ID NO 32 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      primer <400> SEQUENCE: 32cccaggactc cacctcagac c                                               21<210> SEQ ID NO 33 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      primer <400> SEQUENCE: 33actgcgggtg ggaccaagcg ctgccgt                                         27<210> SEQ ID NO 34 <211> LENGTH: 197 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      hNGAL with T7 tag encoded by phNGAL 101 <400> SEQUENCE: 34Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr    50                  55                  60                   Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65                  70                  75                  80  Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp                 85                  90                  95      Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Ser Gln         115                 120                 125             Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly Ser Ala Trp Ser His Pro Gln Phe Met Ala Ser Met Thr Gly             180                 185                 190         Gly Gln Gln Met Gly          195                           <210> SEQ ID NO 35 <211> LENGTH: 178 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      wild type hNGAL <400> SEQUENCE: 35Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65                  70                  75                  80  Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn                 85                  90                  95      Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln         115                 120                 125             Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly  <210> SEQ ID NO 36 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      oligonucleotide <220> FEATURE:  <221> NAME/KEY: modified_base<222> LOCATION: (4)..(9)<223> OTHER INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 36ccannnnnnt gg                                                         12<210> SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      5xHis tag <400> SEQUENCE: 37 His His His His His 1               5    <210> SEQ ID NO 38 <211> LENGTH: 6 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      6xHis tag <400> SEQUENCE: 38 His His His His His His 1               5        <210> SEQ ID NO 39 <211> LENGTH: 79<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      oligonucleotide <220> FEATURE:  <221> NAME/KEY: modified_base<222> LOCATION: (20)..(21)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (26)..(27)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (32)..(33)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (35)..(36)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (47)..(48)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (53)..(54)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (59)..(60)<223> OTHER INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 39caagagctac aatgtcacan nkgtcnnktt tnnknnkaag aagtgtnnkt acnnkatcnn     60kacttttgtt ccaggttcc                                                  79<210> SEQ ID NO 40 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      oligonucleotide <400> SEQUENCE: 40ggtgacattg tagctcttat cttctttcag ctcatagatg gtggc                     45<210> SEQ ID NO 41 <211> LENGTH: 64 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      oligonucleotide <400> SEQUENCE: 41ggagaagcgg atgaagttct cctttagttc cgaagccagc tccttggttc tcccgtagag     60ggtg                                                                  64<210> SEQ ID NO 42 <211> LENGTH: 567 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polynucleotide <220> FEATURE:  <221> NAME/KEY: CDS<222> LOCATION: (1)..(564) <400> SEQUENCE: 42cag gac tcc acc tca gac ctg atc cca gcc cca cct ctg agc aag gtc       48Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val         1               5                   10                  15              cct ctg cag cag aac ttc cag gac aac caa ttc cat ggg aag tgg tat       96Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr                     20                  25                  30                  gtg gta ggt ctc gca ggg aat gca att ctc aga gaa gac aaa gac ccg      144Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro                 35                  40                  45                       caa aag atg tat gcc acc atc tat gag ctg aaa gaa gac aag agc tac      192Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr             50                  55                  60                          aat gtc acc tcc gtc ctg ttt agg aaa aag aag tgt gac tac tgg atc      240Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile         65                  70                  75                  80          agg act ttt gtt cca ggt tcc cag cca ggc gag ttc acg ctg ggc aac      288Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn                         85                  90                  95               att aag agt tac cct gga tta acg agt tac ctc gtc cga gtg gtg agc      336Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser                     100                 105                 110                  acc aac tac aac cag cat gct atg gtg ttc ttc aag aaa gtt tct caa      384Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln                 115                 120                 125                      aac agg gag tac ttc aag atc acc ctc tac ggg aga acc aag gag ctg      432Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu             130                 135                 140                         act tcg gaa cta aag gag aac ttc atc cgc ttc tcc aaa tct ctg ggc      480Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly         145                 150                 155                 160         ctc cct gaa aac cac atc gtc ttc cct gtc cca atc gac cag tgt atc      528Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                         165                 170                 175              gac ggc agc gct tgg tcc cac ccg cag ttc gaa aaa taa                  567Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys                                      180                 185                                      <210> SEQ ID NO 43 <211> LENGTH: 188 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polypeptide <400> SEQUENCE: 43Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1               5                   10                  15      Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr             20                  25                  30          Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro         35                  40                  45              Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr     50                  55                  60                  Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65                  70                  75                  80  Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn                 85                  90                  95      Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser             100                 105                 110         Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln         115                 120                 125             Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu     130                 135                 140                 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145                 150                 155                 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile                 165                 170                 175     Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys             180                 185              <210> SEQ ID NO 44<211> LENGTH: 567 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polynucleotide <400> SEQUENCE: 44ttatttttcg aactgcgggt gggaccaagc gctgccgtcg atacactggt cgattgggac     60agggaagacg atgtggtttt cagggaggcc cagagatttg gagaagcgga tgaagttctc    120ctttagttcc gaagccagct ccttggttct cccgtagagg gtgatcttga agtactccct    180gttttgagaa actttcttga agaacaccat agcatgctgg ttgtagttgg tgctcaccac    240tcggacgagg taactcgtta atccagggta actcttaatg ttgcccagcg tgaactcgcc    300tggctgggaa cctggaacaa aagtcctgat ccagtagtca cacttctttt tcctaaacag    360gacggaggtg acattgtagc tcttgtcttc tttcagctca tagatggtgg catacatctt    420ttgcgggtct ttgtcttctc tgagaattgc attccctgcg agacctacca cataccactt    480cccatggaat tggttgtcct ggaagttctg ctgcagaggg accttgctca gaggtggggc    540tgggatcagg tctgaggtgg agtcctg                                        567<210> SEQ ID NO 45 <211> LENGTH: 404 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polynucleotide <220> FEATURE:  <221> NAME/KEY: CDS<222> LOCATION: (2)..(403) <220> FEATURE:  <221> NAME/KEY: modified_base<222> LOCATION: (32)..(34)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (44)..(49)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (71)..(73)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (80)..(82)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (128)..(130)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (134)..(136)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (140)..(145)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (155)..(157)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (161)..(163)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (167)..(169)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (212)..(214)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (224)..(226)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (233)..(235)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (242)..(244)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (299)..(301)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (305)..(307)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (320)..(322)<223> OTHER INFORMATION: a, c, t, g, unknown or other <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (326)..(328)<223> OTHER INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 45c caa ttc cat ggg aaa tgg tat gtc gtg ggc nnn gcc gga aat nnn nnn     49Gln Phe His Gly Lys Trp Tyr Val Val Gly Xaa Ala Gly Asn Xaa Xaa         1               5                   10                  15              ctg cgt gag gat aag gat ccg nnn aaa atg nnn gcg acc att tac gag       97Leu Arg Glu Asp Lys Asp Pro Xaa Lys Met Xaa Ala Thr Ile Tyr Glu                     20                  25                  30                  ttg aaa gaa gat aaa tca tat aac gtc acc nnn gtg nnn ttt nnn nnn      145Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Xaa Val Xaa Phe Xaa Xaa                 35                  40                  45                      aag aaa tgc nnn tac nnn att nnn acc ttt gtg ccg ggg agc cag ccg      193Lys Lys Cys Xaa Tyr Xaa Ile Xaa Thr Phe Val Pro Gly Ser Gln Pro             50                  55                  60                          ggc gag ttt act tta ggc nnn att aaa agt nnn ccg ggc nnn aca tca      241Gly Glu Phe Thr Leu Gly Xaa Ile Lys Ser Xaa Pro Gly Xaa Thr Ser         65                  70                  75                  80          nnn ttg gtc cgc gtc gtg agc acc aac tac aac cag cat gcc atg gtg      289Xaa Leu Val Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val                         85                  90                  95              ttc ttc aag nnn gtg nnn cag aac cgc gag nnn ttt nnn atc aca ctg      337Phe Phe Lys Xaa Val Xaa Gln Asn Arg Glu Xaa Phe Xaa Ile Thr Leu                     100                 105                 110                 tac ggg cgc acg aaa gaa ctg aca agc gag ctg aag gaa aat ttt atc      385Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile                 115                 120                 125                     cgc ttt tcc aaa tct ctg g                                            404Arg Phe Ser Lys Ser Leu                                                      130                                                                  <210> SEQ ID NO 46 <211> LENGTH: 134 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polypeptide <220> FEATURE:  <221> NAME/KEY: MOD_RES<222> LOCATION: (11)..(11) <223> OTHER INFORMATION: Any amino acid<220> FEATURE:  <221> NAME/KEY: MOD_RES <222> LOCATION: (15)..(15)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (16)..(16)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (24)..(24)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (27)..(27)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (43)..(43)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (45)..(45)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (47)..(47)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (48)..(48)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (52)..(52)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (54)..(54)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (56)..(56)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (71)..(71)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (75)..(75)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (78)..(78)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (81)..(81)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (100)..(100)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (102)..(102)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (107)..(107)<223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (109)..(109)<223> OTHER INFORMATION: Any amino acid <400> SEQUENCE: 46Gln Phe His Gly Lys Trp Tyr Val Val Gly Xaa Ala Gly Asn Xaa Xaa 1               5                   10                  15      Leu Arg Glu Asp Lys Asp Pro Xaa Lys Met Xaa Ala Thr Ile Tyr Glu             20                  25                  30          Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Xaa Val Xaa Phe Xaa Xaa         35                  40                  45              Lys Lys Cys Xaa Tyr Xaa Ile Xaa Thr Phe Val Pro Gly Ser Gln Pro     50                  55                  60                  Gly Glu Phe Thr Leu Gly Xaa Ile Lys Ser Xaa Pro Gly Xaa Thr Ser 65                  70                  75                  80  Xaa Leu Val Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val                 85                  90                  95      Phe Phe Lys Xaa Val Xaa Gln Asn Arg Glu Xaa Phe Xaa Ile Thr Leu             100                 105                 110         Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile         115                 120                 125             Arg Phe Ser Lys Ser Leu      130                  <210> SEQ ID NO 47<211> LENGTH: 404 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polynucleotide <400> SEQUENCE: 47ccagagattt ggaaaagcgg ataaaatttt ccttcagctc gcttgtcagt tctttcgtgc     60gcccgtacag tgtgatctta aagtactcgc ggttctggga cactttcttg aagaacacca    120tggcatgctg gttgtagttg gtgctcacga cgcggaccaa gtatgatgtc aggcccgggt    180aacttttaat gttgcctaaa gtaaactcgc ccggctggct ccccggcaca aaggtacgaa    240tccagtagtc gcatttcttt ttgcgaaaca acacggaggt gacgttatat gatttatctt    300ctttcaactc gtaaatggtc gcatacattt tctgcggatc cttatcctca cgcagaatgg    360catttccggc caggcccacg acataccatt tcccatggaa ttgg                     404<210> SEQ ID NO 48 <211> LENGTH: 197 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic      polypeptide <400> SEQUENCE: 48Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Gln Asp Ser Thr Ser 1               5                   10                  15      Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn             20                  25                  30          Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Leu Ala         35                  40                  45              Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro Gln Lys Met Tyr Ala     50                  55                  60                  Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Ser Val 65                  70                  75                  80  Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile Arg Thr Phe Val Pro                 85                  90                  95      Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn Ile Lys Ser Tyr Pro             100                 105                 110         Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser Thr Asn Tyr Asn Gln         115                 120                 125             His Ala Met Val Phe Phe Lys Lys Val Ser Gln Asn Arg Glu Tyr Phe     130                 135                 140                 Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys 145                 150                 155                 160 Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly Leu Pro Glu Asn His                 165                 170                 175     Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly Ser Ala Trp             180                 185                 190         Ser His Pro Gln Phe          195         

1-78. (canceled)
 79. A method of producing a lipocalin mutein that iscapable of binding hepcidin with an affinity by a KD of about 10 nM orlower, wherein the lipocalin mutein comprises: (i) a set of mutatedamino acid residues at the sequence positions 96, 100, and/or 106 of thelinear polypeptide sequence of mature human neutrophilgelatinase-associated lipocalin (hNGAL), selected from the groupconsisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, andTyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, andTyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn96→Thr, Tyr 100→Val, and Tyr 106→Gly; and (ii) at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acidresidues at any of the sequence positions corresponding to the sequencepositions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127,132, and 134 of the linear polypeptide sequence of mature hNGAL, whereinthe lipocalin mutein is produced starting from the nucleic acid codingfor the lipocalin mutein by means of genetic engineering methods.
 80. Apharmaceutical or diagnostic composition comprising a lipocalin muteinthat is capable of binding hepcidin with an affinity by a KD of about 10nM or lower, wherein the lipocalin mutein comprises: (i) a set ofmutated amino acid residues at the sequence positions 96, 100, and/or106 of the linear polypeptide sequence of mature human neutrophilgelatinase-associated lipocalin (hNGAL), selected from the groupconsisting of (a) Asn 96→Val, Tyr 100→Gln, and Tyr 106→unchanged (b) Asn96→Arg, Tyr 100→Glu, and Tyr 106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, andTyr 106→Gly, (d) Asn 96→Gly, Tyr 100→Gly, and Tyr 106→Gly, (e) Asn96→Lys, Tyr 100→Ala, and Tyr 106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, andTyr 106→Val, (g) Asn 96→Ser, Tyr 100→Val, and Tyr 106→Arg, and (h) Asn96→Thr, Tyr 100→Val, and Tyr 106→Gly; and (ii) at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acidresidues at any of the sequence positions corresponding to the sequencepositions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127,132, and 134 of the linear polypeptide sequence of mature hNGAL, and apharmaceutically acceptable excipient.
 81. A lipocalin mutein thatcomprises an amino acid sequence as set forth in any one of SEQ ID NOs:1-14.
 82. A lipocalin mutein that is capable of binding hepcidin with anaffinity by a KD of about 10 nM or lower, wherein the mutein has atleast 90% sequence identity to an amino acid sequence as set forth inany one of SEQ ID NOs: 1-14.
 83. The lipocalin mutein according to claim81, wherein the lipocalin mutein is fused at its N-terminus and/or itsC-terminus to a fusion partner which is a protein, or a protein domainor a peptide.
 84. The lipocalin mutein according to claim 81, whereinthe mutein is conjugated to a compound that extends the serum half-lifeof the mutein.
 85. A nucleic acid molecule comprising a nucleotidesequence encoding the lipocalin mutein according to claim
 81. 86. Anisolated host cell containing a nucleic acid molecule of claim
 85. 87.The lipocalin mutein according to claim 82, wherein the lipocalin muteinis fused at its N-terminus and/or its C-terminus to a fusion partnerwhich is a protein, or a protein domain or a peptide.
 88. The lipocalinmutein according to claim 82, wherein the mutein is conjugated to acompound that extends the serum half-life of the mutein.
 89. A nucleicacid molecule comprising a nucleotide sequence encoding the lipocalinmutein according to claim
 82. 90. An isolated host cell containing anucleic acid molecule of claim
 89. 91. A method of forming a complexwith hepcidin and inhibiting the ability of hepcidin to bind to orinteract with ferroportin in a subject, comprising the step ofadministrating a lipocalin mutein that is capable of binding hepcidinwith an affinity by a KD of about 10 nM or lower to the subject, whereinthe lipocalin mutein comprises: (i) a set of mutated amino acid residuesat the sequence positions 96, 100, and/or 106 of the linear polypeptidesequence of mature human neutrophil gelatinase-associated lipocalin(hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly,Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser,Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr106→Gly; and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, or 17 mutated amino acid residues at any of the sequencepositions corresponding to the sequence positions 36, 40, 41, 49, 52,68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linearpolypeptide sequence of mature hNGAL.
 92. A method of treatment ordiagnosis of a disease or disorder involving a disorder of ironhomeostasis or an inflammatory condition associated with an elevatedlevel of hepcidin, comprising the step of administering to a subject inneed thereof a lipocalin mutein that is capable of binding hepcidin withan affinity by a KD of about 10 nM or lower to the subject, wherein thelipocalin mutein comprises: (i) a set of mutated amino acid residues atthe sequence positions 96, 100, and/or 106 of the linear polypeptidesequence of mature human neutrophil gelatinase-associated lipocalin(hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly,Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser,Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr106→Gly; and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, or 17 mutated amino acid residues at any of the sequencepositions corresponding to the sequence positions 36, 40, 41, 49, 52,68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linearpolypeptide sequence of mature hNGAL.
 93. A diagnostic or analytical kitcomprising a lipocalin mutein that is capable of binding hepcidin withan affinity by a KD of about 10 nM or lower, wherein the lipocalinmutein comprises: (i) a set of mutated amino acid residues at thesequence positions 96, 100, and/or 106 of the linear polypeptidesequence of mature human neutrophil gelatinase-associated lipocalin(hNGAL), selected from the group consisting of (a) Asn 96→Val, Tyr100→Gln, and Tyr 106→unchanged (b) Asn 96→Arg, Tyr 100→Glu, and Tyr106→Phe, (c) Asn 96→Asp, Tyr 100→Ser, and Tyr 106→Gly, (d) Asn 96→Gly,Tyr 100→Gly, and Tyr 106→Gly, (e) Asn 96→Lys, Tyr 100→Ala, and Tyr106→Ile, (f) Asn 96→Ser, Tyr 100→Arg, and Tyr 106→Val, (g) Asn 96→Ser,Tyr 100→Val, and Tyr 106→Arg, and (h) Asn 96→Thr, Tyr 100→Val, and Tyr106→Gly; and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, or 17 mutated amino acid residues at any of the sequencepositions corresponding to the sequence positions 36, 40, 41, 49, 52,68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linearpolypeptide sequence of mature hNGAL.